Isolation of marine bacteria from sponges in the south central coastal region of vietnam with brown seaweed polysaccharide degrading activities

The potential of producing brown seaweed polysaccharide-degrading enzymes of marine bacteria associated with sponges from Phu Quy Island and Van Phong Bay, Viet Nam was studied on algina

ISOLATION OF MARINE BACTERIA FROM SPONGES IN THE

SOUTH-CENTRAL COASTAL REGION OF VIETNAM WITH

BROWN SEAWEED POLYSACCHARIDE-DEGRADING

ACTIVITIES

Vo Thi DieuTrang*, Cao Thi Thuy Hang, Phan Thi Hoai Trinh, Ngo Thi Duy Ngoc,

Huynh Hoang Nhu Khanh, Tran Thi Thanh Van

Nha Trang Institute of Technology Research and Application, VAST, 02 Hung Vuong,

Nha Trang, Khanh Hoa, Viet Nam

*

Email: vtrang47@gmail.com

Received: 3 September 2020: Accepted for publication: 3 December 2020

Abstract Oligosaccharides, transformed products of seaweed polysaccharides, have been

reported to have valuable biological activities, in which they act as so they are potential compounds for biomaterials, biofuels, as well as nutrients, cosmetic and medical fields Polysaccharide-degrading enzymes are the best tools for the preparation of these bioactive oligosaccharides The potential of producing brown seaweed polysaccharide-degrading enzymes

of marine bacteria associated with sponges from Phu Quy Island and Van Phong Bay, Viet Nam was studied on alginate and fucoidan extracted from brown seaweeds In that, screening for

producing alginate lyase by using alginate prepared from Sargassum mcclurei (S mcc A), and producing fucoidanase by using fucoidan prepared from S mcclurei (S mcc F), S polycystum (S poly F) and S oligocystum (S oli F) as reference substrates The results in the present study

showed that thirty-five bacterial strains were isolated from twenty sponge samples in both positions The proportion of positive isolated bacterial strains on S mcc A, S mcc F, S poly F, and S oli F substrates were 68.6 %, 34.3 %, 48.6 %, and 20 %, respectively Analyzing 16S rRNA sequences of four selected bacterial strains, which exhibited enzymatic activities on a large number of tested polysaccharides, 1911PQ-1.37.6, 1911PQ-1.37.2, 1911VP-3.52.6, and 1911VP-3.28.2 showed substantial similarity (higher 99.5 % of percent identity and 100 %

query cover) with Staphylococcus arlettae, S pasteuri, Bacillus megaterium and Alteromonas macleodii, respectively These investigations demonstrated the potential of bacterial associated

with sponges in the south-central coastal region of Viet Nam Sea for the explorations of novel seaweed polysaccharide-degrading enzymes

Keywords: alginate lyase, bacteria, fucoidanase, seaweed polysaccharides, sponges

Classification numbers: 1.2.1, 1.3.2, 1.5.3

1 INTRODUCTION

Phaeophyceae-class brown seaweeds are the primary source of marine poly saccharides in the marine environment Their polysaccharides with various biological activities have been

applied in various fields of biomaterials, biofuels, food, and cosmetics and have been attracting

more and more attention to study [1] In Viet Nam, the brown seaweeds, especially the

Sargassum genus, commonly grow in the South-central coastal region with particular interesting

compounds in alginate and fucoidans [2]

Alginate is the main polysaccharide derived from brown seaweeds known for containing

three types of block structure: M block (β-L-mannuronate), G block (α-L-guluronate), and M-G

block (containing both poly uronic acids) Depending on the species of brown seaweeds and

their growing conditions, the block structure and the ratio of M/G in alginate molecules vary

significantly [3] Fucoidans, complex fucose-rich polysaccharide, are also extracted from brown

seaweeds They contain fucose, galactose, xylose, mannose, arabinose, uronic acid, sulfate

groups, and acetyl group in their composition Fucoidans have been increasingly studied in

pharmaceutical applications over the years due to wide spectrum of biomedical activities as

anticancer, anticoagulant, antitumor, antioxidant, anti-inflammatory, anti-thrombotic, and

immunomodulatory activity [4] Even though having numerous pharmaceutical properties, the

applications of seaweed polysaccharides like alginate and fucoidan in medicines still have many

restrictions because of their diverse and complex structures [5] Thus, the investigation of the

productions of oligo and monosaccharides by using specific enzymes would solve the

relationship between the structure and function of seaweed poly saccharides and increased their

practical potential values

Marine microorganisms are essentially interesting sources of seaweed

polysaccharide-degrading enzymes with high specificity and work under milk conditions for the synthesis of

oligosaccharides [6] They are isolated from different marine organisms, including seaweeds, sea

urchins, sponges, and sea cucumbers The discovery and characterization based on their

properties have been made focused on the marine bacteria of alginate lyases and fucoidanases

recently Alginate lyase sare characterized into two groups according to their specific block of

substrates: block M (EC 4.2.2.3) and block G (EC 4.2.2.11) [7] Fucoidanases are enzymes that

catalyze the cleavage of the glycoside bonds between sulphated fucosyl residues in fucoidans

substrates According to similarities of amino acid sequences, secondary structures, and cleaved

glycoside bond, fucoidanases are classified into the family 107 of Glycoside Hydrolases

(GH107) in the CAZy database [8]

Although more and more investigations on the alginate lyase and fucoidanase bacterial

have been reported on the world so far, little is known regarding this issue in Viet Nam,

especially in the South-Central coastal region This research aims to screen for new sources of

enzyme among bacteria isolated from sponges collected from Phu Quy and Van Phong Islands

that utilized alginate and fucoidan of brown seaweed Sargassum genus (S mcclurei, S

polycystum, S oligocystum) as well as to identify the potential strains

2 MATERIALS AND METHODS 2.1 Seaweed polysaccharide substrates

Crude polysaccharides from S mcclurei, S polycystum, and S oligocystum were extracted

as described by Zvyagintseva et al [9] Fucoidans were further purified by anion-exchange

chromatography on the DEAE-Macro prep resin column by Thuan et al [10] Alginate from

S mcclurei was extracted as previously described in [9]

2.2 Sample collection

Sponges were collected by scuba from Phu Quy Island (10o30’28”N, 108o56’02”E) and Van Phong Bay (12o34’30”N, 109o23’59”E) at the depth ranging from 5 - 10 m in November

2019 The samples were stored in sterile plastic bags and transported to the laboratory at 4 oC for

bacterial isolation and further experiments

2.3 Isolation of potential seaweed polysaccharide transformation bacteria

In the preliminary experiments, 1 g of collected sponges were ground with 1 ml sterile seawater 100 µl of suspension was spread on marine broth media (M.B.) containing 15g/L agar, 5 g/L peptone, 2 g/L yeast extract, 0.2 g/L K2HPO4 0.05 g/L of MgSO4 and added

with1g/L polysaccharide from S mcclurei as the specific carbon sources of marine bacteria

The plates were incubated at 30 °C and checked daily until colonies were visible with eyes Morphologically different colonies were transferred onto a new plate with on M.B media (without polysaccharide) to obtain pure bacterial cultures The pure isolated bacterial strains were selected and then stocked in sterile M.B media with 40 % glycerol at -80 °C in the Marine Microorganism Collection of Nha Trang Institute of Technology Research and Application

(NITRA)

2.4 Screening marine bacteria for enzymatic activities

The bacterial isolates were screened for the production of alginate lyase by Gram’s

iodine plate method [11], using 1 % alginate from brown seaweed S mcclurei as substrates The

cell growth of isolated bacteria was cultured on alginate-agar plates for 24 h at 30 oC The cultured plates were then removed off their biomass and flooded with Gram’s iodine for 5 to 10 min the appearance of clear zone, visualized after washing under tap water indicated the bacteria secreted enzymes with alginate-modifying activity

The bacterial isolates were screened for the production of fucoidan-modifying

enzymes by the fucoidan-agar plate method as described by Shichenko et al [12] In brief, the isolates were cultivated for 3 days at 28 °C on fucoidan-agar medium containing 1% (w/v) crude fucoidan from S mcclurei, S oligocystum, and S polycystum Bacterial cells were removed from the agar plate surface, and a 1 % aqueous solution of hexadecyl trimethylammonium bromide (cetavlon) was added After incubating for 30 mins at 25 ⁰C, the agar plates were washed with water several times Transparent areas under the colonies indicated bacteria-secreted enzymes with fucoidan-modifying activity

2.5 Identification of the selected bacteria

Strains exhibiting high polysaccharide-degrading enzyme activities (as determined using fucoidan/alginate-agar plate method) were identified based on the analysis of 16S ribosomal RNA (rRNA) gene sequence Cells from 5 mL overnight culture in M.B liquid media were collected by centrifugation, and genomic DNA of selected strains was isolated

by DNeasy Blood and Tissue DNA kit (Qiagen), following manufacturer’s recommendations for Gram-positive bacteria The 16S rRNA gene fragment was amplified using Phusion®High-Fidelity DNA Polymerase (NEB, U.S.) with the universal bacterial primers (533F: 5’-GTGCCAGCAGCCGCGGTAA3’ and 1392R: 5’-GGTTACCTTGTT-ACGACTT-3’), checked with PCR product on 2 % of agarose gel This product was then

purified with a QIA quick PCR purification kit (Qiagen) [13] The purified 16S rRNA was

sequenced on the ABIT 3130XL system (Thermo Fisher Scientific, USA) at Nam Khoa

Company, Ho Chi Minh city Sequences were then aligned against 16S rRNA entries of National Center for Biotechnology Information (NCBI) reference sequence database to identify the taxonomic identity of the closest bacterial homologs Then, their 16SrRNA sequence data were also submitted to Genbank for registering Genbank Accession numbers

2.6 Phylogenetic analysis

The 16S rRNA nucleotide sequence of the closest bacterial neighbors with the selected strains were identified by comparing with those in GenBank database using the Basic local alignment search tool for nucleotide (BLASTn) Sequences were aligned using the multiple sequence alignment tool in CLC Main Workbench 8 program The phylogenetic tree was constructed with Kimura model, as a neighbor joining tree and 1000 of bootstrap value [14]

RESULTS AND DISCUSSION 3.1 Isolation of potential seaweed polysaccharide transformation bacteria

Bacteria, which grow up on media containing various carbohydrate compounds, would have to produce necessary primary metabolites like enzymes to hydrolase these compounds, then absorb nutrient ingredients for their life In the primary screening, the used media contained

polysaccharide compounds extracted from S mcclurei as carbon sources, which had a high

amount of sulphated polysaccharide (fucoidan) and alginic acid

1911PQ-1.07.2

1911PQ-1.13.1

1911PQ-1.13.2

1911PQ-1.20.1

1911PQ-1.34.1

1911PQ-1.34.2

1911PQ-1.34.4

1911PQ-1.37.1

1911PQ-1.37.2

1911PQ-1.37.3

1911PQ-1.37.4

1911PQ-1.37.6

1911VP-3.04.1

1911VP-3.24.1

1911VP-3.24.2

1911VP-3.28.1

1911VP-3.28.5

1911VP-3.52.6

Figure 1.Colony characteristics of marine bacteria isolates from sponges collected at Phu Quy and

Van Phong Islands

Therefore, the growth bacteria isolated from this media were expected as fucoidanases and alginate lyase producers The results of the isolation showed that 35 strains of aerobic bacteria

were isolated from 20 sponge samples at both collected sites Indeed, 21 strains were isolated from 13 sponge samples in Phu Quy Island, and 14 strains were isolated from 07 sponge samples

in Van Phong Bay The isolated bacterial strains had different colony morphological characteristics with a variety of colors, sizes, and shapes on solid MB medium after 24 hour-incubation at 30 °C, morphologies of representative isolates are shown in Figure 1 The number

of isolated strains grown on media containing S mcclurei polysaccharides as carbon sources

meant that sponges would be an abundant source of marine organisms for discovering marine polysaccharide degrading enzymes, especially fucoidanase and alginate lyase

3.2 The abilities to produce seaweed polysaccharide-degrading enzymes of the isolated bacteria

In marine ecosystems, polysaccharides from seaweeds are a rich carbon source for different organisms, and it is likely that such organisms, e.g invertebrates, bacteria and fungi, would be able to degrade the algal polysaccharides Bacteria are one of the essential drivers of carbon cycling across ecosystems where they secrete enzymes that breakdown complex polysaccharides and release short oligosaccharides For bacteria, the processing of alginate and fucoidan require polysaccharide-degrading enzymes such as alginate lyase and fucoidanase Almost all other studies focus on discovering alginate lyase/fucoidanase from bacteria isolated from seaweeds or gut of seaweed feeders [15].In this study, we look for bacterial candidates that isolated from sponges in Vietnam Sea where there are abundant polysaccharide sources of alginate and fucoidan specifically from brown seaweeds

Figure 2.The ability to produce alginate lyase and fucoidanase of bacterial isolated from sponges in the

South-Central coastal region of Vietnam Sea

To evaluate the abilities to degrade seaweed polysaccharides of isolated bacteria, alginate

from S mcclurei and fucoidan from S mcclurei, S polycystum and S oligocystum were used as

specific substrates for activations of the isolated strains Figure 2 illustrated the proportion of bacterial strains active on different substrates Figure 3 showed activities of isolated strains on S mmc A and S oli F substrates by the alginate and fucoidan-containing media plate assays, respectively

0 20 40 60 80 100

68.6

34.3

48.6

20

Seaweed polysaccharide substrates

The most significant proportion (68.6%) of the isolated bacteria demonstrated alginate utilization, which is showed by the light brownish clearance zone around agar lytic colonies after staining Gram’s iodine (Figure 2) The active zones from 04 to 18 mm showed different alginate lyase producing capacity of bacterial isolates (Figure 3a) A similar trend also came with the

investigation by Nguyen Thi Thuan, who reported that about 59.79% of bacterial strains isolated

from sponge showed alginate lyase activity [16] The content of alginate from brown seaweed,

especially the 20-40% high from S mcclurei from which the abundant source of carbon those

isolates prioritize using for their life [15], might explain the high proportion of alginate lyase

producing bacteria in this study Therefore, this result from our study agreed with the previous

report that besides seaweeds and soft corals, sponges would be the potential marine organisms for the screening of alginate lyase producing bacteria

Figure 3.Activities of isolated strains on S mmc A and S oli F substrates by the alginate and

fucoidan-containing media plate assay (a): The alginate lyase producing activities showed on the diameter (mm) of the light brownish clearance zone (b): The fucoidanase producing activities showed on the transparent zones with “-”: precipitated area, “+”: transparent zone but not clear,“++”: transparent and clear

zone,“+++”: transparent and very clear zone

In this investigation, the isolated strains also showed significant differences in proportions

of activities on S mcc F, S poly F, and S oli F with 34.3%, 48.6%, and 20%, respectively (Figure 2).These differences could be related to the different structural characteristics of fucoidan extracted from various seaweeds, especially monosaccharide compositions and

linkages of the main backbone Fucoidan extracted from Sargassum species as S mcclurei, S polycystum, and S oligocystum belong to the most structurally diverse fucoidan group with the

varying ratio of monosaccharides components, glycoside bonds, and sulphated groups, etc They are sulphated galactofucan with fucose and galactose as the major sugar components in their backbone or their branches The minor constituents of glucose, xylose, mannose, or rhamnose have also been observed The sulphate groups are present at C2 and/or C4 of fucosyl residues The main backbone of these fucoidans has been proposed to diversity consisting of α(1→3)-linked L-fucosyls, α(1→4)-α(1→3)-linked L-fucosyl, as well as β(1→3) linkages from galactosyl to fucosyl or α(1→6) linkages from fucosyl to galactosyl, etc depending on species of seaweeds

[17]

Most of the isolated strain showed activities on S poly F ,which was reported to contain

many monosaccharides composition like fucose, galactose, glucose, xylose, mannose, rhamnose

and differ linkages in the backbone like α(1→3)-linked L-fucosyls, β(1→3/4)-linked D-galactose, β(2→3)-linked D-mannose/xylose [18] The diverse and complicated structure of

fucoidan from S polycystum helped enzymes of the isolated bacteria active easier than on the

others Indeed, only 20 % of isolated strains showed active on S oli F that had a less

heterogeneous monosaccharide composition (only containing fucose, galactose, and mannose),

with sulphate content of 35 % and not contain acetates [19] The exciting results were that most

of these enzymes exhibited high activities within the clear or very clear transparent zone on this substrate (Figure 3b) It could be explained that the limits of the structure of S oli F contained the specific linkages for enzyme activations Meanwhile, S mcc F collected at Nha Trang bay, Viet Nam, also to be a unique galactofucan structural moieties with sulphated α(1→3) L-fucosyl and α(1→4) linked galactosyl residues The fucosyl residues in S mcc F are sulphated at C2 and/or at C4, and some of the galactosyl moieties are sulphated at C6 [17] The activation of

isolated bacteria on alginate and fucoidan also extracted from S mcclurei indicated that this

brown seaweed would be potential substrates to screening and studying the seaweed polysaccharide-degrading enzyme in the future

3.3 The identifications of selected strains with the potential of producing seaweed polysaccharide-degrading enzyme

Table 1.Capacities of alginate lyase and fucoidanase activities selected isolated bacterial

Selected

bacterial

strains

Morphological

characteristics of

selected strains

Sources of isolates

Alginate lyase activities (mm)

Fucoidanase activities

S mm A S mm

F

S poly

F S oli F

Note: “-” No activity detected on fucoidan agar plates; “+” Low activity on fucoidan agar plates;

“++” Medium activity on fucoidan agar plates; “+++” High activity on fucoidan agar plates

Base on the results of screenings of bacteria producing fucoidanase and alginate lyase, we investigated four potential strains with two strains (1911PQ-1.37.2 and 1911PQ-1.37.6) having

the highest activities on fucoidan from S oligocystum, and two strains (1911VP-3.28.2 and

1911VP-3.52.6) having broad spectra activities on alginate from S mcclurei and fucoidan from

S mcclurei and S polycystum Their activities and characterizations were further details in Table 1

As mentioned above, S oli F has quite a simple structure with high amounts of sulphate

content (35 %) and only containing fucose, galactose, and minus of mannose in their main backbone Hence, 1911PQ-1.37.2 and 1911PQ-1.37.6 that showed the highest activities on S oli

F would expect catalytic cleavage linkage of this substrate Despite this, we need more works to

illustrate the detailed structure of fucoidan from S oligocystum to indicate the unique catalysis

of fucoidanase obtained from these selected strains The degradation on S mmc A, 1911PQ-1.37.2 and 1911PQ-1.37.6 showed a significant difference with positive (12 mm) and negative results, respectively The data obtained from here likely reflects the diversity of capacities between the produced enzymes, especially alginate lyase from these strains even when they are associated with the same source

Figure 4 Phylogenetic tree of 04 selected bacterial strains with potential producing seaweed

polysaccharide for degrading enzyme The tree was constructed from a comparison of 16S rRNA gene sequence using the neighbor-joining analysis of distance matrix with Kimura model Bootstrap values (expressed as percentages of 100 replications) more than 75 % are shown at branch points

Methanocaldococcus jannaschii DSM 2661 (accession number NR_074233.1) was used as outgroup of

strains The scale bar represents 0.15 substitutions per nucleotide position

The selected strains of 1911VP-3.28.2 and 1911VP-3.52.6 did not exhibit enzyme activities

on S oli F but showed on all of the other substrates like S mm A, S mm F, and S poly F, which were known to contain difference linkages in their backbone Therefore, they were expected to produce the enzymes that could be used as multiple tools to degrading polysaccharide with containing α(1→3)-linked L-fucosyls, β(1→3/4)-linked galactose, β(2→3)-linked D-mannose/xylose in the backbone of S poly F or sulphated α(1→3) L-fucosyl and α(1→4) linked galactose residues in the backbone of S mmc F

More useful information can be collected by determining habitat life and characterizing strains, in which, 16S rRNA genes sequences of the 04 selected strains that exhibited high potential producing seaweed polysaccharide and degrading enzyme were sequenced, classified

Phylum Firmicutes

Phylum Proteobacteria

and subsequently aligned to construct a phylogenetic tree (Figure 4) Each identified selected strains were submitted on NCBI and registered to Genbank accession number

The analysis indicated that the selected strain 1911PQ-1.37.2, 1911PQ-1.37.6, 1911VP-3.28.2,and 1911VP-3.52.6 showed 99.62 %, 99.8% , 99.61 %, and 100 % sequence similar with

Staphylococcus pasteuri(JQ267500.1), S arlettae (MN851077.1), Alteromonas macleodii (JQ267500.1), and Bacillus megaterium (FJ174644.1), respectively They were named as S pasteuri 1911PQ-1.37.2, S arlettae 1911PQ-1.37.6, A macleodii 1911VP-3.28.2, and B megaterium 1911VP-3.52.6 with accession number MT669360, MT669343, MT669369, and

MT669368, respectively Three selected strains are gram-positive bacteria and belonging to the

phylum Firmicutes while A.macleodii 1911VP-3.28.2 is gram-negative and belonging to the phylum Proteobacteria (Figure 4) B megateriumwas recorded as a popular species from Nha

Trang Bay in Tran Nguyen Ha Vy’s publication [20] and reported to produce high alginate-lyase

activity on alginate extracted from brown seaweed S mcclurei [16] In this study, the strain of B megaterium1911VP-3.52.6 were isolated in Van Phong Bay, this strain was not only producing alginate-lyase on S mcclurei alginate but also fucoidanase on S mcclurei and S polycystum

fucoidan substrates Although there are a number of publications on finding new bacterial strains for seaweed polysaccharide degrading enzymes, however this number are still limited compared

to potential availability Thus, these identified bacterial strains would be considered as a primarily source for exploring polysaccharide-degrading enzymes from marine bacteria

4 CONCLUSION

The investigated results in our study illustrated the potential of producing seaweed polysaccharide-degrading enzymes derived from bacteria isolated from sponges in the South-Central coastal region of Vietnam Sea Thirty-five potential strains were isolated from 21 sponge samples collected at Phu Quy and Van Phong Islands All of them were found to be

polysaccharide-degrading enzyme producers with 68.6 % strains producing alginate lyase on S mcclurei algiante; 34.3 %, 48.6 %, and 20 % strains producing fucoidanase on S mcclurei, S polycystum and S oligocystum fucoidan substrates, respectively Four selected strains were identified to belong to three different families (Staphylococcaceae, Alteromonadaceae, and Bacillaceae) in the scientific classification of Bacteria Kingdom, which in turn demonstrated the diversity of the selected strains Among them, two identified strains S pasteuri 1911PQ-1.37.2 and S arlettae 1911PQ-1.37.6 showed significant activities on fucoidan extracted from S oligocystum, which proved themselves as that would be novel bacteria strains for producing

specific enzyme The data obtained here provide valid foundations that may aid in the advanced research for seaweed polysaccharide degrading enzyme and reference substrates

Acknowledgment: The research funding from Vietnam National Foundation for Science and

Technology Development (NAFOSTED-106.02-2018.353) was acknowledged

CRediTauthorship contribution statement: Vo Thi DieuTrang: Methodology, Experiments, Analysis and

Supervision Cao Thi Thuy Hang: Methodology, Analysis, and Supervision Phan Thi Hoai Trinh: Experiments Ngo Thi Duy Ngoc: Experiments Huynh Hoang Nhu Khanh: Methodology Tran Thi Thanh Van: Methodology, Analysis, Supervision and Funding acquisition

Declaration of competing conflict: The authors declare that there is no conflict of interest

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