Accepted Manuscript Title: Exopolysaccharide production by a marine Pseudoalteromonas sp. strain isolated from Madeira Archipelago ocean sediments Author: Christophe Roca Mareen Lehmann Cristiana A.V. Torres S´ılvia Baptista Susana P. Gaudˆencio Filomena Freitas Maria A.M. Reis PII: DOI: Reference:

S1871-6784(16)00018-2 http://dx.doi.org/doi:10.1016/j.nbt.2016.02.005 NBT 862

To appear in: Received date: Revised date: Accepted date:

4-11-2015 21-1-2016 16-2-2016

Please cite this article as: Roca, C., Lehmann, M., Torres, C.A.V., Baptista, S., Gaudˆencio, S.P., Freitas, F., Reis, M.A.M.,Exopolysaccharide production by a marine Pseudoalteromonas sp. strain isolated from Madeira Archipelago ocean sediments, New Biotechnology (2016), http://dx.doi.org/10.1016/j.nbt.2016.02.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Highlights  Isolation of a Pseudoalteromonas sp. strain from Madeira Archipelago ocean sediments  The strain presents autolytic behavior during batch cultivation

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 Synthesis of EPS with high uronic acids content

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 Fermentation broth develops high viscosity at the end of cultivation

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Exopolysaccharide production by a marine Pseudoalteromonas sp. strain isolated from

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Madeira Archipelago ocean sediments

Christophe Roca1, Mareen Lehmann1, Cristiana A.V. Torres1, Sílvia Baptista1, , Susana P.

UCIBIO-REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia,

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Gaudêncio1,2, Filomena Freitas1, Maria A.M. Reis1

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Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.

LAQV-REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia,

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Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.

Corresponding Author: Christophe Roca, UCIBIO, REQUIMTE, Departamento de Química,

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Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica,

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Portugal. Phone: +351 212948385. Email: [email protected]

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Abstract Exopolysaccharides (EPS) are polymers excreted by some microorganisms with interesting properties and used in many industrial applications. A new Pseudoalteromonas sp. strain,

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MD12-642, was isolated from marine sediments and cultivated in bioreactor in saline culture medium containing glucose as carbon source. Its ability to produce EPS under saline

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conditions was demonstrated reaching an EPS production of 4.4 g/L within 17 hours of

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cultivation, corresponding to a volumetric productivity of 0.25 g/L.h, the highest value so far obtained for Pseudoalteromonas sp. strains. The compositional analysis of the EPS revealed

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the presence of galacturonic acid (41-42 mol%), glucuronic acid (25-26 mol%), rhamnose (16-22 mol%) and glucosamine (12-16 mol%) sugar residues. The polymer presents a high

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molecular weight (above 1,000 kDa). These results encourage the biotechnological

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exploitation of strain MD12-642 for the production of valuable EPS with unique

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composition, using saline by-products/wastes as feedstocks.

Keywords: Marine sediments; Pseudoalteromonas; Extracellular polysaccharide (EPS); Autoinhibition.

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Introduction Exopolysaccharides (EPS) are polymers excreted by some microorganisms as a protective barrier against harmful conditions. Many microbial EPS, such as xanthan or gellan gums,

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isolated from terrestrial sources are being successfully exploited in several industries. Indeed, EPS can be used in a wide range of biotechnological applications, such as thickening agents,

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stabilizers and texturizers in the food industry, flocculating agents in the wastewater

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treatment industry, or anti-aging molecules in the cosmetics industry [1, 2].

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Marine environments are highly complex habitats that present extremely variable conditions of temperature, salinity and pressure. It is therefore to be expected that bacteria isolated from

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these environments have developed different adaptation mechanisms compared to terrestrial ones, including for example the synthesis of exopolysaccharides (EPS) with distinctive and

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diverse composition that allowed their survival. This diversity is of great interest as it offers the possibility to discover completely new molecules with unique features and properties,

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such as anti-bacterial, algaecide or anti-fouling activities [3, 4, 5]. The world´s oceans are still

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overlooked and rather unexploited, remaining an open and very promising research field for EPS discovery [6]. Undoubtedly, the production of innovative marine EPS, with growing biotechnological applications for several industrial sectors, presents a huge added-value. To date, three main genera of marine EPS-producing bacteria have been identified: Pseudoalteromonas sp., Alteromonas sp. and Vibrio sp., producing polymers with original structures and good product yields, ranging from 0.5 to 4.0 g/L during cultivation on glucose [7]. These strains are usually isolated from extreme environments, such as deep sea hydrothermal vents [8]. There are very few commercially available EPS produced specifically by marine bacteria [9, 10]. One such example is Hyalurift, a hyaluronic acid-like 4 Page 4 of 33

EPS produced by the deep sea bacterium Vibrio diabolicus [11], commercialized by SeadevFermenSys and Infremer, presenting great potential with proven capacities of in vivo regeneration of tissues, such as bone and skin, and the ability to accelerate in vitro collagen fibrillation and activate fibroblasts [12].

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The genus Pseudoalteromonas, belonging to the class Gammaproteobacteria, shows a widespread distribution in the marine environment [13], being mostly associated with marine

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eukaryotic hosts. Pseudoalteromonas strains have been found to produce many active

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metabolites and a wide range of EPS [14]. Chemical composition and molecular weight revealed that these were very diverse, even among closely related Pseudoalteromonas

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isolates. Most of the EPS contain charged uronic acid residues; several also present sulphate groups. Here, we report the isolation of a new Pseudoalteromonas sp. strain from marine

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sediments samples collected in shallow waters off the Madeira Archipelago and demonstrate

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its ability to synthesize large amounts of EPS under saline conditions, using marine culture medium. The produced biopolymer was characterized in terms of composition and average

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molecular weight. The potential biotechnological exploitation of this strain to produce EPS

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from saline feedstocks is discussed.

Materials and Methods

Sample collection and processing Sediment samples were collected in the Macaronesia Atlantic ecoregion, off the Madeira Archipelago, in the Southern reaches of Madeira and Porto Santo Islands and in the Western reach of Desertas Islands. The shallow sediments were collected by scuba diving from depths of 10-20 m. A modified sediment sampler (Kahlsico, El Cajon, CA, model #214WA110) was used to sample the remaining sediments to depths of 1.310 m. The samples consisted of 662 5 Page 5 of 33

sediments, alternating from fine muds to small rocks and small pieces of dead coral. Each sample was transferred to a labelled sterile bag (Nasco whirl-pack) and immediately stored on ice for transportation to shore. Long-term storage was at -20 °C. The samples were processed using the following heat-shock and drying methods:

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- Method 1 (M1): wet sediment (c. 0.5 g) was diluted with 2 mL of sterile seawater (SSW). After mixing, the diluted sample was allowed to settle for a few minutes, heated to 55 oC for

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6 min. 100 µl of the top layer was spread on an agar plate;

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- Method 2 (M2): wet sediment was diluted into 2 ml of SSW (dilutions 1:2), heated to 55 oC for 6 min, and 50 µl of the resulting solution was spread on an agar plate;

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- Method 3 (M3): a sterile cotton plug (1-2 cm in diameter) was used to repeatedly stamp previously dried sediment (overnight in a laminar flow chamber) onto the surface of an agar

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plate in a circular direction.

A total of 198 sediment samples were processed within a few hours of collection using M1

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and M2. All 662 samples were processed one month after collection using M3. The samples were processed and inoculated as described above onto the surface of one to three of the

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following agar media (per litre):

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- Medium 1 (A1): 18 g agar, 10 g starch, 4 g yeast extract, 2 g peptone; - Medium 2 (1/2 A1): 18 g agar, 5 g starch, 2 g yeast extract, 1 g of peptone; - Medium 3 (SSW): 18 g agar.

All media were prepared with natural seawater collected on site and deionized water in the proportion 75:25 (v/v), containing the anti-fungal agent cycloheximide (20 mg/mL).

Unicellular bacteria quantification and isolation Inoculated Petri dishes were incubated at RT (c. 25-28oC) and monitored periodically over 6 months for unicellular bacteria growth. Bacteria were visually quantified on each plate by

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counting of colony forming units. Special attention was given to colonies with shiny and slimy morphology that were indicative of the strains’ ability for producing viscous EPS. Seven hundred and forty four colonies were successively transferred onto new media until obtention of pure cultures. All pure 744 strains were grown in liquid culture (medium 1

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without agar) and cryopreserved in 10% (v/v) glycerol at -80oC

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Screening of EPS producing capacity

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From the 744 isolated strains, 500 were selected for screening of their EPS production capacity in shake flask cultivation, based on their slimy aspect. The strains were cultivated in

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25 mL shake flasks on liquid medium containing per litre (750 mL sea water and 250 mL deionized water): 30 g glucose, 4 g yeast extract and 2 g peptone. Starch was replaced by

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glucose to facilitate polymer purification and avoid possible interference with viscosity

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200 rpm.

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measurement. The cultures were incubated in an orbital shaker, during 5 days, at 28 ºC and

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DNA extraction, 16S rRNA gene amplification and phylogenetic analyses Taxonomic identification of a selection of producing strains was performed. The strains were cultured in 4 mL of medium A1, at 200 rpm and 25 °C for 3 days. The Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA), for Gram negative bacteria, was used, as described in the Wizard® Genomic DNA Purification Kit Technical Manual, #TM050. The 16S rRNA gene was polymerase chain reaction (PCR) amplified using the primers FC27 (5´-AGAGTTTGATCCTGGCTCAG-3´)

and

RC1492

(5´-

TACGGCTACCTTGTTACGACTT-3´) and the products purified using SureClean PCR cleanup kit (BioLine, London, UK), using the protocol provided by the manufacturer.

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Sequences were obtained using the primers listed above at STABVIDA, Portugal (http://www.stabvida.net/), using ABI BigDye® Terminator v3.1 Cycle Sequencing Kit and purified. Purified products were run on an ABI PRISM® 3730xl Genetic Analyzer and

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sequence traces were edited using Sequencing Analysis 5.3.1 from Applied Biosystems.

Phylogenetic analysis

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The forward and reverse 16S rRNA sequences obtained were checked for accurate base

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calling using BioEdit Sequence Aligment Editor version 7.2.0 [15] and assembled and analyzed using BLAST (Basic Local Alignment Search Tool) [16], available on the NCBI

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website (http://www.ncbi.nlm.nih.gov/). Sequence was aligned using Clustal X version 2 [17], and imported into Mesquite version 2.75 [18] for manual alignment and masking. A

Bioreactor experiments

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version 2 with 1,000 bootstrap replicates.

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neighbor-joining phylogenetic tree was constructed from 1,630 base pairs using the Clustal X

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The selected MD12-642 strain was cultivated under controlled conditions in a 2 L bioreactor

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(BioStat B-plus, Sartorius), with a working volume of 1.75 L. Medium composition was the same as for the screening procedure. The aeration rate was maintained at 2 SLPM (standard liter per minute) during the cultivation. The temperature was controlled at 30 ± 0.1 °C and the pH was controlled at 7.0 ± 0.05 by the automatic addition of NaOH (5 M).

Analytical methods Culture growth was followed by measuring the optical absorbance of the broth at 550 nm. The viscosity of the culture broth samples was measured using a digital viscometer (Brookfield Engineering Laboratories Inc., USA), at RT. Glucose concentration in the

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fermentation broth was determined by high-performance liquid chromatography (HPLC) using a Metacarb 87H column (Varian) and a refractive index detector (RI-71, Merck). The column was eluted at 30 ºC with 0.01 N H2SO4 solution at a flow rate of 0.5 mL/min.

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EPS extraction

The EPS was recovered from the cultivation broth as described by Freitas et al. [19]. Briefly,

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the culture broth samples were centrifuged (13,000×g, 15 min). Bacterial enzymes

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responsible for polymer degradation were inactivated by heat treatment for 1 h at 70 ºC. Residual cell debris and protein precipitates were removed from heat-treated supernatants by

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centrifugation (13,000×g, 15 min). Removal of low molecular weight compounds was performed using dialysis membranes (SnakeSkinTM, Thermo Scientific) with a 10,000 Da

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molecular weight cut-off, against deionized water (72 h, 4 ºC). Dialysed supernatants were

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EPS characterization

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lyophilised.

The lyophilized EPS was analysed for its sugar composition and acyl groups composition, as

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well as the content in contaminants reminiscent from the culture broth, namely, inorganic

salts and proteins. For the analysis of sugar composition, dried EPS samples (∼5 mg) were

dissolved in 5 mL deionized water and hydrolyzed with trifluoroacetic acid (TFA) (0.1 mL TFA 99%) at 120 °C, for 2 h. The hydrolysate was used for the quantification of the constituent monosaccharide monomers by HPLC, using a CarboPac PA10 column (Dionex), 9 Page 9 of 33

equipped with an amperometric detector. The analysis was performed at 30 °C, with NaOH 4 mM as eluent, at a flow rate of 0.9 mL/min. The acyl groups were quantified in the acid hydrolysate by HPLC with an Aminex HPX-87H column (BioRad), coupled to an UV

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detector, using H2SO4 0.01 N as eluent, at 50 °C and a flow rate of 0.6 ml/min.

The total inorganic content of the EPS samples was evaluated by subjecting them to pyrolysis

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at a temperature of 550 °C for 48 h. The total protein content was determined by a modified

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Lowry method. EPS aqueous solutions (4.5 g/L) samples (5.5 mL) were mixed with 1 mL 20% (w/v) NaOH, placed at 100 °C for 5 minutes and cooled on ice. Each sample was mixed

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with 170 μL of CuSO4·5H2O (25%, v/v) and centrifuged (3500 × g, 5 min). The optical density was measured at 560 nm (Spectrophotometer Helios Alpha, Thermo Spectronic, UK).

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Albumin (Merck) solutions (0.5–3.0 g/l) were used as protein standards. The EPS average molecular weight (Mw) was determined by SEC-MALLS (Size Exclusion

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Chromatography – Multi-Angle Laser Light Scattering). SEC was performed at 35 °C using a HPLC System (Agilent Infinity1260) operating at isocratic conditions at a flow rate of

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1 mL/min. As mobile phase 0.1 M LiNO3 was used. The samples (1g/L) were dissolved in

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the eluent directly and stirred overnight followed by a centrifugation at 14,000 x g for 5 min. An injection volume of 100 µL in combination with a set of three columns (2 x PSS Suprema 10,000 Å, 1 x PSS Suprema 100 Å (8 x 300 mm; 10 µm) and a pre-column) were used for the analysis. The elution was monitored by a refractive index detector (Agilent G1362A) and a multi angle light scattering detector (Brookhaven SLD 7000). Twelve different pullulan standards between 342 Da to 2.56 MDa were used to perform the calibration. Data acquisition and molecular weight calculations were performed by the PSS WinGPC UniChrom software.

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Results and Discussion

Isolation and identification of MD12-642 strain From a total of 500 strains screened for EPS production, out of 744 bacterial colonies

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obtained from sediments collected at 15 m depth off the Madeira Archipelago, around 8% were considered as good EPS producers, with production greater than 1.0 g/L (Table 1). The

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remaining collection presented little or no EPS production (< 1.0 g/L), under the tested

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conditions. The highest producers (> 2.5 g/L) represented 2% of the screened strains (Table

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1).

Strain description was based on identification through 16S rRNA gene sequencing, and

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showed that the isolates included representatives of the genera Pseudoalteromonas, Alteromonas, Psychrobacter, and Brevibacterium (Table 2). Interestingly, most of the

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producing strains that were identified belonged to the Pseudoalteromonas genus (Table 2). Members of this genus are generally found in eukaryotic hosts, associated with marine

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animals (i.e. tunicate and mussels) or marine algae. To date, Pseudoalteromonas contains 43

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species and 885 non-characterized strains that have been isolated from several different living and non-living marine sources, widely distributed in the marine environment [13]. In particular, the MD12-642 strain was isolated from a marine sediment sample collected at 15 m, at Desertas Islands (lat. 32 35.346; long. 16 33.060). Morphologically it is a Gramnegative straight rod, motile with a single polar flagellum. On plates, it forms nonpigmented transparent colonies. The 16S rRNA gene sequencing analysis revealed 99 % homology with Pseudoalteromonas hodoensis, with a close relationship within other non-pigmented members of the Pseudoalteromonas genus, as represented in Figure 1 (GenBank accession number for MD12-642 16S rRNA sequence: KT828543). Even though EPS production is a 11 Page 11 of 33

common trait within Pseudoalteromonas strains (Table 1) as a way of enhancing survival in extreme marine environments, such as hydrothermal vents [14, 20], very few have been studied in detail. None of the closest neighbours (including P. hodoensis) of MD12-642 have been documented as EPS producers, suggesting that Pseudoalteromonas MD12-642 is a

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novel EPS producer.

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Bioreactor cultivation of strain MD12-642

Batch cultivation of the MD12-642 was performed in a controlled bioreactor to understand

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better and improve EPS production. Although this strain was not the highest EPS producer,

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the concentration of EPS produced in 25 mL flasks nevertheless reached around 2.0 g/L and MD12-642 was the only strain whose EPS production was improved during bioreactor

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cultivation and was therefore selected for further studies. Figure 2 presents the cultivation profile of MD12-642 growing on glucose using 75% (v/v) sea water under batch conditions.

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During the exponential growth phase, glucose was consumed in less than 12 h and MD12642 presented a specific growth rate as high as 0.60 h-1. Once glucose was depleted, the

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optical density (OD) of the broth dropped abruptly, probably because of cell lysis. Actually,

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some marine strains of Pseudoalteromonas species, such as P. tunicata, P. citrea and P. rubra, have already been found to display autoinhibitory activity [3]. For instance, P. tunicata has been reported to produce an autocidal protein during biofilm growth, resulting in partial cell death, suggesting ecological advantages to the species by generating a metabolically active subpopulation of dispersal cells [21]. This might be the case of Pseudoalteromonas MD12-642, which apparently autolyzes just after glucose is depleted (Figure 2). In order to identify a putative trigger of MD12-642 autolysis, similar batch cultivation was performed (as previously described in Figure 2), but this time, a 15 g/L glucose pulse was 12 Page 12 of 33

added after 6.5 h (Figure 3). In this way, the cells entered stationary phase maintaining their cell density to OD ~13, without lysing. Only once glucose was depleted (~20 h), the OD decreased dramatically, clearly demonstrating that a limitation in carbon can result in cell autolysis.

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EPS production by strain MD12-642

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In the batch experiment, an EPS production of 2.5 g/L was observed by the time glucose was exhausted (~15 h) (Figure 2), corresponding to a maximum volumetric productivity of 0.17

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g/L.h (Table 3). This value is higher than those reported in the literature for other marine

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Pseudoalteromonas, Zunongwangia or Alteromonas EPS-producing strains [7, 22] (Table 3). For instance, a productivity of 0.06 g/L.h was achieved in a 7 days cultivation of a

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Pseudoalteromonas strain isolated from a sponge sample from the Red Sea [23]. Zunongwangia profunda SM-A87 isolated from deep-sea sediment can secrete large

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quantities of EPS, when grown on lactose, peptone and yeast extract (8.90 g/L), but after 8 days, corresponding to a productivity of 0.05 g/L.h [24]. Only the marine Pantoea sp. BM39

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strain presented higher performances, with 21 g EPS/L within 18 h cultivation on glucose,

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corresponding to a productivity of 1.2 g/L.h [25]. The EPS production observed after glucose depletion might have been due to the consumption of other medium components, namely, yeast extract and peptone, which may have served as additional carbon sources for the culture. On the other hand, upon cell lysis, high molecular weight intracellular and cell membrane components (e.g. proteins, nucleic acids, etc.) may have been solubilized in the cultivation broth and may have been carried over with the synthesized EPS during the extraction procedure. This may have contributed to the overestimation of the polysaccharide fraction.

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In the pulse-fed experiment (Figure 3), a maximum EPS production of 4.4 g/L was obtained at 17 hours of cultivation. This corresponds to a volumetric productivity of 0.25 g/L.h, which is higher than the value obtained in the batch experiment. These results show that EPS productivity by Pseudoalteromonas sp. MD12-642 can be improved by avoiding glucose

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depletion. Contrary to the batch experiment (Figure 2), in the pulse-fed experiment, no EPS synthesis was detected after glucose depletion and the concomitant cell lysis. This may have

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been due to the fact that, in the batch experiment, the culture was under substrate limiting

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conditions for a longer period of time (~27 h), while in the pulse-fed experiment that period was shorter (~16 h) (Figures 2 and 3). Hence, the solubilization of cell components upon cell

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lysis may have been less extensive in the pulse-fed experiment and, thus, a lower amount of high molecular weight components were carried over with the polysaccharide fraction during

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extraction.

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Concomitant with EPS production, the fermentation broth viscosity increased dramatically in both experiments. In the pulse-fed experiment, the apparent viscosity of the broth increased

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from around 1.0 cP, at the beginning of the cultivation, to more than 10,000 cP at the end of the batch cultivation (measured at a shear rate of 0.05 s-1) (Figure 4). For the same shear rate,

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this is equivalent to the viscosity of a 0.2% aqueous solution of xanthan, one of the most popular thickening gums in the food industry (cpkelco.com). Moreover, the broth developed a shear thinning behavior, with the apparent viscosity decreasing as the shear rate increased (Figure 4). Most of the viscosity build-up was observed up to 15 h of cultivation, which corresponds to the time prior to glucose depletion and cell lysis. This suggests that the observed broth viscosity was due to the EPS synthesized by Pseudoalteromonas sp. MD12642. Afterwards, the viscosity still increased by an order of magnitude, which was no longer related to EPS synthesis that maintained the same concentration between 17 and 35 h of cultivation (Figure 4). Therefore, this viscosity increase may have been due to the 14 Page 14 of 33

solubilisation of cell components resulting from cell lysis or to the EPS molecules adopting a different conformation influenced by the changing broth composition. Viscosity build-up during fermentation is a common characteristic to marine EPS producing bacteria. Alteromonas macleodii isolated from a deep sea hydrothermal shrimp was found to

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produce viscous exopolysaccharide [26]. However, to the best of our knowledge, values in the ranges of the one obtained with Pseudoalteromonas sp. MD12-642 have not been

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documented. Only, the strain Zunongwangia profunda SM-A87, isolated from deep sea

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sediments was found to reach a fermentation broth viscosity of 6,500 cP [24].

EPS characterization

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The EPS recovered from the cell free supernatant presented protein and inorganic salts contents of 13 and 7% (w/w), respectively, which shows that the sample was not completely

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purified. Acyl groups represented 23% (w/w) of the recovered EPS dry mass, including acetate (13%), succinate (9%) and pyruvate (1%). Sugar monomer composition of the

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extracted EPS were evaluated and the presence of galacturonic acid (41 – 42 mol%),

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glucuronic acid (25 – 26 mol%), rhamnose (16 -22 mol%) and glucosamine (12 – 16 mol%) was detected (Table 4). Traces of glucose, mannose and galactose were also identified but, as they are common monomers present in yeast extract and were detected in the cultivation medium prior to inoculation, they were not considered as polymer components, but rather as contaminants. Sugar monomer composition of the EPS samples remained quite stable between batch and pulse-fed experiments, before and after cell autolysis (Table 4). The production of polysaccharides by Pseudoalteromonas strains IAM, SM9913 and AM mainly rich in glucose has been reported by several authors [23, 27, 28]. Bai et al. [29] reported the production of an EPS composed of mannose, glucose and galactose by strain S15 Page 15 of 33

5. Pseudoalteromonas SM20310 produced a polymer composed of mannose, glucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and xylose [30]. As far as the authors know, this is the first report of an EPS composed of glucuronic acid, glucosamine, galacturonic acid and rhamnose being synthesized by a Pseudoalteromonas sp. Moreover, the

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presence of uronic acids in such high content, especially glucuronic acid, may render the EPS interesting properties for biotechnological uses. Glucuronic acid-based polysaccharides, such

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as hyaluronic acid and heparin, have proven biomedical applications in surgery, regenerative

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medicine, tissue engineering and as active ingredients in anti-thrombotic and anti-arthritic drugs [31]. The molecular weight determined by SEC-MALLS revealed a polymer with high

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molecular weight, above 106 Da. Future work will focus on attempting a better characterization of the polymer in terms of structural analysis, which will involve defining

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the downstream procedures necessary to obtain pure polysaccharide fractions. In addition, attention will be paid to the utilization of saline industrial wastes, such as fish cannery

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processing waters. These effluents are usually difficult to treat biologically because of their high salinity and their potential as feedstock to produce as EPS has never been fully

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exploited. Given that strain MD12-642 was isolated in medium containing sea water, it will

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be easily adapted to these salty feedstocks, which opens new opportunities to explore marine resources for biotechnology processes, namely in the biopolymer area.

Conclusion

The new strain Pseudoalteromonas sp. MD12-642 isolated from Madeira Archipelago ocean sediments was shown to synthesize EPS with unique composition in bioreactor cultivation. The culture was able to produce EPS with high molecular weight and high productivity under saline culture conditions. The presence of rare sugars as constituents of the EPS, together 16 Page 16 of 33

with its capacity to confer high viscosity to the broth, makes this process interesting for further innovative development in particular for the fish/marine industry generating saline waste streams, which could be used as feedstocks.

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Conflict of interests

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The authors declare no conflict of interests.

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Acknowledgements

(FCT)

through

Projects

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This research is the result of financial support from Fundação para a Ciência e a Tecnologia PTDC/QUI-QUI/119116/2010,

UID/QUI/50006/2013

and

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UID/Multi/04378/2013, and the European Union 7th Framework Programme (FP7/20072013) under grant agreement n° PCOFUND-GA-2009-246542. F. Freitas and C. V. Torres

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acknowledge FCT fellowships SFRH/BPD/87774/2012 and SFRH/BPD/72280/2010,

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respectively. The authors thank W. Fenical, P. R. Jensen and C. A. Kauffman from Scripps Institution of Oceanography, CA, USA, for all the support given to perform the sampling collection. P. Castilho from Universidade da Madeira and M. Freitas from Estação de Biologia Marinha, Funchal, Portugal, are also acknowledged for the logistic support provided during the sampling expedition. We thank I. S-Sanches from UCIBIO REQUIMTE-FCTUNL for the assistance regarding the taxonomic identification. We thank Broder Rühmann and Jochen Schmid from Technische Universität München, Lehrstuhl für Chemie Biogener Rohstoffe, Schulgasse, Straubing, Germany, for their assistance in SEC analysis. We also

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thank the German Academic Exchange Service – DAAD for supporting cooperation between

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UCIBIO and TUM.

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[32] Skovhus TL, Holmstrom C, Kjelleberg S, Dahllof I. Molecular investigation of the distribution, abundance and diversity of the genus Pseudoalteromonas in marine samples. FEMS Microbiol. Ecol, 2007;61: 348–361. [33] Sutherland IW. Biotechnology of Microbial Exopolysaccharides, Cambridge Studies

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List of Figures Figure 1: Neighbour-joining phylogenetic tree of Pseudoalteromonas strains closest to

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the MD12-642 strain based on 1,000 bootstrap replicates.

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Figure 2: Glucose consumption, growth and EPS production during batch cultivation of

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Pseudoalteromonas sp. MD12-642 in 2 L bioreactor

Figure 3: Glucose consumption, growth and EPS production during batch cultivation of

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Pseudoalteromonas sp. MD12-642 in 2 L bioreactor with a pulse of glucose.

Figure 4: Flow curves for culture broth samples at different cultivation times (measured at

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30◦C) during pulse-fed experiment

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List of Tables

Table 1: Shake flask EPS production by marine bacteria isolated from Madeira Archipelago

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ocean sediments.

Table 2: Taxonomic identification of higher yield marine bacteria EPS producers, isolated

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from Madeira Archipelago ocean sediments.

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Table 3: Marine bacteria EPS production studies comparison.

Table 4: Monosaccharide profile (in % mol) of the EPS produced by the isolated

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Pseudoalteromonas strain MD12-642 during batch cultivation and pulse feeding.

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EPS (g/L) 1.87 1.70 1.67 1.63 1.55 1.5 1.46 1.42 1.4 1.31 1.29 1.14 1.12 1.12 1.09 1.07 1.05 1.05 1.03 0.99

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Strain code MD12-517 MD12-506 133 MD12-365 MD12-540 36-2 MD12-331a 34 501 MD12-331b 145 212 MD12-518 210 MD12-115 94 MD12-574 MD12-555 MD12-085 349

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EPS (g/L) 6.88 6.08 5.67 4.74 3.60 3.32 3.22 2.92 2.85 2.79 2.61 2.39 2.15 2.09 2.05 2.03 2.02 2.01 2.00 1.95

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Strain code MD12-540 MD12-423 MD12-130 MD12-375 MD12-030 MD12-515 MD12-506 MD12-03 122 MD12-523 MD12-085 MD12-642 MD12-640 MD12-528 MD12-512 MD12-375 158 468 MD12-523 36

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Homology EPS (g/L) (% ) 99 2.39 MD12-642 Pseudoalteromonas hodoensis 87 1.29 145 Pseudoalteromonas prydzensis 98 2.01 468 Pseudoalteromonas atlantica 98 1.39 501 Pseudoalteromonas mariniglutinosa 99 0.78 MD12-530 B Psychrobacter submarinus 99 0.74 MD12-044 1/2 Bacillus licheniformis 99 0.73 MD12-236 c Bacillus subtilis 97 1.46 MD12-331 A Pseudoalteromonas atlantica 99 0.66 MD12-331 B Brevibacterium halotolerans 99 4.75 MD12-375 Pseudoalteromonas shioyasakiensis 99 6.08 MD12-423 SWA Bacillus subtilis subsp. Inaquosorum 91 1.05 MD12-574 B Bacillus sonorensis a bacteria were identified throught gene 16S sequencing by their closest match obtained from BLAST. Bacterium(a)

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Strain code

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ip t cr Carbon source

Culture method

Glucose

Pseudoalteromonas sp. MD12-642 Pseudoalteromonas ruthenica

Ocean sediments from Madeira Archipelago Sea water next to atomic power plant

Pseudoalteromonas sp. AM

Sponge from Red Sea

Glucose

Pseudoalteromonas SM20301

Arctic Sea Ice

Glucose

Pseudoalteromonas sp. S.5

Antarctic Sea Ice

Productivity (g/L.h) 0.17 0.25

Shake flask

72 h

1.80

0.025

[33]

7 days

9.50

0.06

[23]

3 days

0.50

0.007

[30]

60 h

0.05

0.0008

[29]

192 h

8.90

0.05

[24]

72 h

9.00

0.125

[32]

d

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Glucose

Batch bioreactor Pulse-fed bioreactor

Cultivati EPS on time (g/L) 15 h 2.50 17 h 4.40

ep te

Glucose Lactose Glucose

Shake flasks, 25 ºC Shake flasks, 15 ºC Shake flasks, 8 ºC Shake flask, 10 ºC Bioreactor, 25ºC

Reference This study

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Zunongwangia profunda SMDeep-sea sediment A87 Alteromonas macleodii Deep-sea hydrothermal MS907 vent shrimp

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Location

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Strain

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Batch Pulse feeding before cell lysis end of cultivation (42 h) before cell lysis end of cultivation (35 h) GlcA 28 25 41 26 GlcN 14 12 13 16 GalA 44 41 22 42 Rha 15 22 25 16 Gal A: galacturonic acid, GlcA: glucuronic acid, Rha: rhamnose, GlcN: glucosamine.

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Monosaccharide

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Figure

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Figure

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Figure

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Figure

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