International Journal of Microbiology 191 (2014) 36–44

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Exploring the diversity of extremely halophilic in food-grade

Olivier Henriet, Jeanne Fourmentin, Bruno Delincé, Jacques Mahillon ⁎

Laboratory of Food and Environmental Microbiology, Université catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium article info abstract

Article history: is one of the oldest means of food preservation: adding decreases activity and inhibits microbial Received 28 April 2014 development. However, salt is also a source of living and archaea. The occurrence and diversity of viable Received in revised form 7 August 2014 archaea in this extreme environment were assessed in 26 food-grade salts from worldwide origin by cultivation Accepted 13 August 2014 on four culture media. Additionally, metagenomic analysis of 16S rRNA gene was performed on nine salts. Viable Available online 20 August 2014 archaea were observed in 14 salts and colony counts reached more than 105 CFU per gram in three salts. All fi Keywords: archaeal isolates identi ed by 16S rRNA gene sequencing belonged to the family and were Food-grade salt related to 17 distinct genera among which , and were the most represented. Halophilic archaea High-throughput sequencing generated extremely different profiles for each salt. Four of them contained a single High salinity media major (Halorubrum, Halonotius or Haloarcula) while the others had three or more genera of similar occurrence. The number of distinct genera per salt ranged from 21 to 27. Halorubrum had a significant contribution to the archaeal diversity in seven salts; this correlates with its frequent occurrence in crystallization ponds. On the contrary, walsbyi, the halophilic archaea most commonly found in solar , was a minor actor of the food-grade salt diversity. Our results indicate that the occurrence and diversity of viable halophilic archaea in salt can be important, while their fate in the gastrointestinal tract after ingestion remains largely unknown. © 2014 Elsevier B.V. All rights reserved.

1. Introduction applications and road de-icing account for 66% and 29%, respectively (The European Salt Producers' Association, 2004). Salt ( , NaCl) is an ingredient extensively used in food Solar salterns and aquatic environments with salinities at or near satu- preparations. Its history stretches back to antiquity, probably coinciding ration have been widely studied as they maintain high microbial densities with the discovery of agriculture and the early means of food preservation (107–108 cells ml−1) due to the lack of predation and high con- (Desrosier, 1970). The use of salt in the food and drink industry has three centration (Oren, 2002). These ecosystems have revealed the joint presence main roles. First, it gives a specific flavor to the product, enhances and mod- of bacteria and archaea, the latter largely dominated by members of the ifies the flavor of other ingredients and reduces the sensation of bitterness. Halobacteriaceae family (Benlloch et al., 2002; Oren, 2002). Currently (as Second, it helps the handling and processing of many products: it makes of April 2014), 40 genera and 158 make up this family whose gluten more stable and less extensible in bread; it regulates the activity of type strains have been isolated from different environmental sources: starter culture in cheese, increases water-holding capacity water and sediment from hypersaline lakes (39%), water from solar salterns and improves the tenderness of meat. Third, and most importantly, salt acts (22%), soils and rocks (16%), food (15%) and other sources (8%). as a food preservative by reducing water activity (Hutton, 2002). Metagenomic assessments of archaeal diversity in solar salterns Large-scale processes of salt production include rock , from different regions of the globe have often reported the presence solution mining, solar evaporation and evaporation with heating of the Halorubrum and Haloquadratum genera (Burns et al., 2004; Oh devices. At small scale, local extraction of salt is a common practice in et al., 2010; Pašić et al., 2005; Zafrilla et al., 2010), while culture-based rural areas where salt sediments are easily accessed (shallow salt methods have identified Haloarcula, Halobacterium, Halorubrum and lakes or saline soil crusts). Worldwide production is led by China as main genera (Benlloch et al., 2001; Oren, 2002; Burns (24%), Europe (21%) and USA (17%) (The European Salt Producers' et al., 2004; Birbir et al., 2007; Bidle et al., 2005). This apparent contra- Association, 2004). Salt applications for food and feed represent 3% diction might be explained by the difficulty to cultivate Haloquadratum of the European consumption whereas industrial or chemical walsbyi, as opposed to the “rapid” colony formation of Haloarcula, Halobacterium and Haloferax species (Birbir et al., 2007; Burns et al., ⁎ Corresponding author at: Laboratory of Food and Environmental Microbiology, Earth 2004; Pašić et al., 2005; Rodríguez-Valera et al., 1998). and Life Institute, Université catholique de Louvain, Croix du Sud, 2-L7.05.12, B-1348 Louvain-la-Neuve, Belgium. Tel.: +32 10 47 33 70. Salted and fermented food products often contain halophilic E-mail address: [email protected] (J. Mahillon). archaea. Denaturing gradient gel electrophoresis revealed the presence

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.08.019 0168-1605/© 2014 Elsevier B.V. All rights reserved. O. Henriet et al. / International Journal of Food Microbiology 191 (2014) 36–44 37

of Halorubrum and species in during of cooling to about 80 °C, 2 ml of filter-sterilized CaCl2 0.5 M was sterilely table olives (Abriouel et al., 2011). Histamine-degrading archaea added to each bottle. The medium was slowly shaken to avoid bubbles belonging to genera Halobacterium and were reported in and poured into petri dishes. salted-fermented fishery products and Haloarcula marismortui was isolated from salted anchovies (Moschetti et al., 2006; Tapingkae et al., 2.2.3. Chemically defined medium (CDM) with pyruvate 2010). Kimchi, a Korean fermented food, was shown to maintain The preparation of solid CDM medium with pyruvate was modified archaeal population of the , , , from Kauri et al. (1990). It was prepared by dissolving 12.5% NaCl,

Halobiforma and genera (Chang et al., 2008). This dish is 5.0% MgCl2·6H2O, 0.5% K2SO4 and 0.03% CaCl2·2H2O in ultrapure prepared with jeotgal, a salted-fermented seafood ingredient from water. The pH was adjusted to 7.5 with Tris–HCl 1 M. Volumes of which five new species of the Halobacteriaceae family were isolated: 250 ml were dispensed in 500 ml bottles and 1.5% unwashed agar Natronococcus jeotgali, Halakalicoccus jeotgali, Haloterrigena jeotgali, (MP Biomedicals) were added. Bottles were placed in a water bath at cibarius and Halorubrum cibi (Roh and Bae, 2009; Roh 95 °C until complete dissolution of agar, then autoclaved. When cooled et al., 2007a, 2007b, 2009, 2010). to about 60 °C, each bottle was supplemented with 1.25 ml autoclaved

The salt itself has proven to contain viable microbial cells. In fact, the NH4Cl 1 M, 0.50 ml autoclaved phosphate buffer at pH 7.5, 0.25 ml filter- first food product from which a member of the Halobacteriaceae family sterilized trace element solution SL4 and 5 ml filter-sterilized sodium was isolated, was a food-grade salt from Trapani, Sicily (Petter, 1931). pyruvate 25% (Carl Roth GmbH, Karlsruhe, Germany). Phosphate buffer

Halorubrum trapanicum was originally known as Halobacterium was prepared by adding appropriate volume of 0.5 M K2HPO4 to 30 ml trapanicum and was renamed in the course of a rearrangement of the of 0.5 M KH2PO4 until reaching pH 7.5. Solution SL4 was prepared by of the Halobacteriaceae (McGenity and Grant, 1995). dissolving in ultrapure water acidified with a few drops of concentrated

To our knowledge, no studies have investigated the occurrence and HCl 36 mg MnCl2·4H2O, 44 mg ZnSO4·7H2O, 230 mg FeSO4·7H2Oand diversity of archaea in food-grade salts. Therefore, this study aims to 5mgCuSO4·5H2O. After gentle hand-shaking, CDM medium was assess the archaeal diversity in 26 food-grade salts from worldwide poured into petri dishes. origin: Europe, Asia, North and South America, Africa and Oceania, by both culture-dependent and independent approaches. 2.2.4. DBCM2 medium DBCM2 medium preparation was modified from Dyall-Smith 2. Materials and methods (2009). SW 25% was prepared by mixing 833 ml SW 30% with 167 ml ultrapure water. Volumes of 250 ml were dispensed into 500 ml bottles 2.1. Food-grade salt samples and 1.5% unwashed agar (MP Biomedicals) were added. The bottles were placed in a water bath at 95 °C until complete dissolution of A selection of 26 food-grade salts was analyzed in this study agar, then autoclaved. 2.5 ml autoclaved Tris–HCl 1 M pH 7.4, 1.25 ml

(Table 1). Their geographical origins cover many regions of the globe autoclaved NH4Cl 1 M, 0.5 ml autoclaved phosphate buffer pH 7.4, (Fig. 1). Visual aspect reveals an important diversity of color and particle 0.25 ml filter-sterilized SL10 solution and 1.1 ml filter-sterilized 25% size (Supplementary Fig. S1). Salts were purchased directly from the sodium pyruvate were pipetted in each bottle. After gentle hand-shaking, producer, from local markets or from Belgian supermarkets and organic DBCM2 medium was poured into petri dishes. food e-markets (Supplementary Table S1). They were stored at room temperature in a dark and dry place. 2.3. Plating

2.2. Culture media Three samples of 1.67 g were taken aseptically from different zones of a pack of commercial food-grade salt, placed in a single 50 ml tube Four media developed to promote archaeal growth were used: and dissolved in 25 ml sterile SW 12% at 30 °C and 120 RPM until MGM, Hv-YPC, DBCM2 and CDM with pyruvate. Their composition complete dissolution of salts (approximately 30 min). Plates were and preparation are described below. inoculated with 250 μl solution and sealed with plastic paraffin film. Incubation was performed at 37 °C for 5 weeks. For each food-grade 2.2.1. Modified growth medium (MGM) salt and each medium, 3 replicates were analyzed with up to 3 dilutions. Solid MGM at a salt concentration of 23% was modified from Dyall- Dilutions with a number of colonies between 15 and 250 were kept for Smith (2009). A stock solution of salt water (SW) 30% was prepared colony forming units (CFU) counting. Plates with less than 15 CFU as previously described (Porter et al., 2005). 767 ml SW 30% was without dilution were counted and plates with more than 250 CFU mixed with 200 ml ultrapure water, 0.5% peptone (Oxoid Ltd., were re-inoculated with higher dilution. Counts are expressed as Basingstoke, Hampshire, UK), 0.1% yeast extract (Oxoid Ltd.) and 1 ml log10 (number of CFU). The sample arithmetic mean is calculated on trace element solution SL10 (Widdel et al., 1983). After complete the 3 replicates and the confidence interval is defined with a Student's dissolution, the pH was adjusted dropwise to 7.5 with Tris base 1 M. t-value calculated with n − 1 degrees of freedom and α = 0.05. Pheno- The solution was topped up to 1 l with ultrapure water and volumes typically distinct colonies were picked, streaked and stored at −80 °C in of 250 ml were dispensed in 500 ml bottles in which 1.5% unwashed MGM 23% with (70:30). agar (MP Biomedicals, Illkirch, France) were added. The bottles were placed in a water bath at 95 °C until complete dissolution of agar. The 2.4. Amplification of 16S rRNA gene from archaeal isolates and sequence medium was autoclaved and poured hot into petri dishes. analysis

2.2.2. Hv-YPC Total DNA from haloarchaeal isolates was extracted by osmotic Solid medium Hv-YPC preparation was modified from Dyall-Smith disruption in ultrapure water as follows: a small amount of colony (2009). YPC 10× was prepared by dissolving 5.0% yeast extract (Oxoid from streaked plates was taken with a pipette tip, placed in 200 μl Ltd.), 1.0% peptone (Oxoid Ltd.), 1.0% casamino acids (Difco Laboratories, ultrapure water and vortex mixed for 1 min. In a PCR tube, 2 μlofthis Le Pont de Claix, France) and 17.6 ml KOH 1 M in ultrapure water to a suspension was mixed with 18 μl PCR mix composed of Colorless

final volume of 1 l. 100 ml ultrapure water, 200 ml SW 30%, 1.5% GoTaq® Flexi Buffer (Promega, Madison, WI, USA), 1.5 mM MgCl2 unwashed agar (MP Biomedicals) and 30 ml YPC 10× were dispensed (Promega), 200 μM deoxynucleoside triphosphates, 0.03 u/μlGoTaq® in 500 ml bottles. The bottles were placed in a water bath at 95 °C until DNA polymerase (Promega), 0.25 mM forward primer S-D-Arch-0007- complete dissolution of agar and the medium was autoclaved. After a-S-19 (5′-TTCCGGTTGATCCYGCCRG-3′)(Massana et al., 1997) and 38 O. Henriet et al. / International Journal of Food Microbiology 191 (2014) 36–44

Table 1 Origin and features of the 26 food-grade salts used in this study. Unless specified, all salts are not iodized and contain no additives. The third column indicates if viable archaeal cells were recovered (+) or not (−)onsolidmedia(Fig. 2). Supplementary information can be found on the web page of the producer and/or importer of the salts (Supplementary Table S1).

Name Salt source and country of origin Recovery of viable archaea on solid media

Alae red salt salt mixed with red volcanic clay, Hawaii, USA − Black Black Sea, Turkey + Bolivian salt Uyuni salt flats, Potosí Department, Bolivia + Camargue salt Salterns, Camargue Region, France + Chardonnay salt , stored in Chardonnay wine oak barrels, California, USA + salt Dead Sea, Israel − German rock salt Rock salt mined from a deposit at a depth of 400–750 m below ground, Central Germany − Gozo Island salt Salterns, Marsalforn, Gozo Island, Malta + Guérande salt Salterns, Guérande, Pays de la Loire, France + Hâlen Môn salt Sea salt, Isle of Anglesey, Wales, United + Hâlen Môn salt, smoked Sea salt infused with smoke from Welsh oak chippings, Isle of Anglesey, Wales, United Kingdom − Himalayan pink salt Rock salt with high iron content from Himalayan plateau + Jozo® salt, iodized Refined iodized sea salt from unspecified origin − Jozo® salt, uniodized Refined sea salt from unspecified origin − La Baleine coarse sea salt Coarse iodized sea salt from unspecified origin + La Palma Flor de Sal Salterns, Fuencaliente Salt Works, Canary Islands, Spain + Lac Rose salt Lac Rose, Senegal + Maras salt Salterns filled with salty from a spring in Maras, Cuzco Region, Peru + Marsel® fine salt Refined iodized sea salt from unspecified origin − Murray River salt Salterns filled with Murray–Darling Basin underground brines, Australia − Nigari salt Magnesium salt, Pacific , Japan − Pakistani rock salt Rock salt crystals from Himalaya mountain range, Pakistan + Palm Island black salt Sea salt blended with activated charcoal, Moloka'i Island, Hawaii, USA − Persian blue rock salt Rock salt from mines located in Semnan, Alborz mountains, Iran + Salish salt Sea salt smoked over red alder from Washington state, USA − Sicilian mountain salt Rock salt crystals from mines in Sicilian mountains, Italy −

reverse primer S-D-Arch-1492-a-A-22 (5′-TACGGYTACCTTGTTACGACTT- Sequences were deposited in the NCBI database under GenBank 3′)(Lane, 1991). Thermal cycling parameters were 2 min at 94 °C, then accession numbers KM257939 to KM258099. 35 cycles of 1 min at 94 °C, 1 min at 57.5 °C and 2 min at 72 °C, and finally 10 min at 72 °C. Ultrapure water was used as negative control. Gel elec- 2.5. DNA extraction from food-grade salts trophoresis was performed at 100 V for 28 min on 0.8% agarose gel in Tris–Acetate–EDTA buffer to confirm the presence of amplicons. Microbial DNA extraction from salts has been optimized in this Sequencing was realized on ABI 3730 XL DNA Analyzer (Life study. Camargue salt was used as model. The extraction steps tested Technologies, Carlsbad, CA, USA) at Macrogen Europe (Amsterdam, in this trial were the i) dissolution of salt (water or SW 12%), ii) pre- The Netherlands). Sequences were matched online against the Ribosomal filtration on 5 μm filter (presence or absence), iii) method of cell con- Database Project Release 11.1 (Cole et al., 2014). centration (centrifugation or filtration), and iv) lysis (in ultrapure

10,11 7 21 9 4 2 5 22 18 17 8 20 13 6 12 19 1 14

15 3 16

Fig. 1. Geographical location of 22 food-grade salts with specified origin. Four salts had an unspecified origin: Jozo® iodized, Jozo® uniodized, La Baleine coarse sea and Marsel® fine salts. 1: Alae red; 2: Black Sea; 3: Bolivian; 4: Camargue; 5: Chardonnay; 6: Dead Sea; 7: German rock; 8: Gozo Island; 9: Guérande; 10: Hâlen Môn; 11: Hâlen Môn smoked; 12: Himalayan pink; 13: La Palma Flor de Sal; 14: Lac Rose; 15: Maras; 16: Murray River; 17: Nigari magnesium; 18: Pakistani rock; 19: Palm Island black; 20: Persian blue rock; 21: Salish; 22: Sicilian mountain. O. Henriet et al. / International Journal of Food Microbiology 191 (2014) 36–44 39 water or a solution containing SDS and proteinase K) and DNA recovery S-D-Arch-0915-a-A-20 primer (5′-GTGCTCCCCCGCCAATTCCT-3′) (phenol/chloroform purification and isopentanol precipitation or (Stahl and Amann, 1991). Amplifications were performed in 25 μl NucleoSpin® Food kit (Macherey-Nagel GmbH, Düren, Germany)). In reactions with Qiagen HotStar Taq master mix (Qiagen Inc., Valencia, addition, all 26 salts were tested with PowerSoil® DNA isolation kit CA, USA), 1 μlofeach5μM primer and 1 μl of template. Reactions (Mo-Bio Laboratories Inc., Carlsbad, CA, USA) as the soil content of unre- were performed on ABI Veriti thermocyclers (Applied Biosytems, fined salts may be significant and hinder PCR reactions. Carlsbad, CA, USA) under the following thermal profile: 95 °C for A sample of 8.0 g food-grade salt was dissolved either in 50 ml SW 12% 5 min, then 35 cycles of 94 °C for 30 s, 54 °C for 40 s, 72 °C for or in ultrapure water at 30 °C and 120 RPM until complete dissolution. 1 min, followed by 1 cycle of 72 °C for 10 min and 4 °C hold. Cellulose acetate filters with a 0.2 μmand5.0μmporesize(Macherey– Ampli fication products were visualized with eGels (Life Technolo- Nagel) were autoclaved and placed on sterile 50 ml syringes. The solution gies, Grand Island, NY, USA). Products were then pooled equimolar was either filtered through a 5.0 μmporesizefilter or not. As microbial and each pool was cleaned with Diffinity RapidTip (Diffinity Genomics, content in food-grade salt is low, cells were concentrated by centrifuga- West Henrietta, NY, USA) and size selected using Agencourt AMPure XP tion (2 min at 10,000 ×g and 4 °C), or by filtering through a filter with a (BeckmanCoulter, Indianapolis, IN, USA) following Roche 454 protocols 0.2 μm pore size. Cell lysis and DNA recovery were achieved either with (454 Life Sciences, Branford, CT, USA). Size selected pools were then the NucleoSpin® Food kit following manufacturer's instructions or with quantified and 150 ng of DNA was hybridized to Dynabeads M-270 a lysis followed by isopentanol precipitation. Briefly, the filter was placed (Life Technologies) to create single-stranded DNA (ssDNA) following in a microcentrifuge with 570 μlTris–EDTA solution. After 5 min in an Roche 454 protocols (454 Life Sciences). ssDNA was diluted and used ultrasonic bath, it was incubated at 37 °C for 15 min, then 30 μl SDS 10% in emPCR reactions, which were performed and subsequently enriched. and 3 μl proteinase K (20 mg/ml) were added and the microcentrifuge Sequencing was performed following established manufacture protocols was incubated at 37 °C for 1 h. For the water lysis, all the previous steps (454 Life Sciences). were replaced by simply pipetting 570 μl ultrapure water with 30 μl Data were quality trimmed, clustered at a 4% divergence using SDS (10%) and incubating the microcentrifuge at 100 °C for 15 min to USEARCH application (Edgar, 2010), checked for chimeras with denature . UCHIME (Edgar et al., 2011) and denoised. Sequences were matched Both protocols were followed by the removal of the filter and the ad- against a database derived from NCBI using a BLASTn+ algorithm dition of 100 μl NaCl 5 M and 80 μl CTAB/NaCl. The solution was incubat- (KrakenBLAST, http://www.krakenblast.com/)(Dowd et al., 2008). Se- ed 10 min at 65 °C. DNA was purified with two phenol/chloroform quences with identity scores to well-characterized database sequences extractions and precipitated with equal volume of isopentanol over- higher than 97% were resolved at the species level, between 95 and night at −20 °C. DNA was pelleted by centrifuging for 15 min at 97% at the genus level, between 90 and 95% at the family level, between 13,000 RPM, rinsed with 500 μl cold ethanol 70% and resuspended in 85 and 90% at the order level, and between 80 and 85% at the class level. 40 μlTris–EDTA buffer. The sequence data have been submitted to the NCBI Sequence Read Archive (SRA) (http://www.ncbi.nlm.nih.gov/sra) under the accession 2.6. Metagenomic analysis number SRP044738.

High-throughput sequencing was performed on 9 food-grade salts 3. Results from various origins to assess their archaeal diversity and relative abun- dance. A nested PCR was performed with two archaeal specificprimer Food-grade salt is a common ingredient of food preparations. Several pairs to prepare adapter-ligated DNA fragments. The first amplification archaea from the family Halobacteriaceae were previously isolated from used primers 340F and 1000R (Gantner et al., 2011). In a PCR tube, 10 μl this extreme environment. However, the occurrence and diversity of of DNA extract was mixed with 90 μl PCR mix composed of Colorless halophilic archaea are still unknown. A combined use of culture-based

GoTaq® Flexi Buffer (Promega), 1.5 mM MgCl2 (Promega), 200 μM and metagenomics approaches were adopted in this study to provide deoxynucleoside triphosphates, 0.03 u/μl GoTaq® DNA polymerase a snapshot of the haloarchaeal content in 26 salts from worldwide (Promega), 0.25 mM forward primer 340F S-D-Arch-0340-a-S-18 (5′- origin. CCCTAYGGGGYGCASCAG-3′) and reverse primer 1000R S-D-Arch- 1043-a-A-18 (5′-GGCCATGCACYWCYTCTC-3′)(Gantner et al., 2011). 3.1. Culture of halophilic archaea on solid media Thermal cycling parameters were 2 min at 95 °C, then 35 cycles of 0.5 min at 95 °C, 0.5 min at 57 °C and 1.5 min at 72 °C, and finally The salinities of the solid media were chosen to offer a large range of 10 min at 72 °C. Ultrapure water was used as negative control and growth conditions: 18% for CDM, 20% for Hv-YPC, 23% for MGM and 25% haloarchaeal isolate as positive control. The success of amplification for DBCM2. Additionally, these media differ in the substrate and trace was confirmed by gel electrophoresis at 100 V for 28 min on 0.8% aga- element compositions. Hv-YPC is the richest medium with yeast extract, rose gel in Tris–Acetate–EDTA buffer. As the amplicon concentration peptone and casamino acids but is not supplemented with trace was low in the PCR product, a concentration step was subsequently per- elements. MGM contains less yeast extract but more peptone, plus 8 formed. For each DNA extract, 3 × 100 μl of PCR products was adsorbed trace elements. Pyruvate is the sole carbon source of the last two on silica-membrane spin column from GenElute™ PCR Clean-Up kit media although they diverge in the number of trace elements (8 for (Sigma-Aldrich Co., St. Louis, MO, USA) following the manufacturer's in- DBCM2 and 4 for CDM). structions, and then eluted with 40 μl Tris–EDTA buffer. DNA concentra- The 26 food-grade salts selected for this study included 3 refined and tion was measured with a NanoDrop ND-1000 spectrophotometer 23 unrefined salts (Table 1). As shown in Fig. 2, the count of viable mi- (NanoDrop Technologies Inc., Wilmington, DE, USA) and normalized croorganisms after 5 weeks of incubation revealed important variations with ultrapure water to a final concentration of 20 ng/μl. Samples among the food-grade salts. Archaeal colonies were observed in 14 salts. were sent to the Research and Testing Laboratory, Lubbock (TX, USA) The visual absence of growth was confirmed by a second incubation pe- for archaeal 16S rRNA gene tag-encoded FLX amplicon pyrosequencing riod of another 5 weeks to ensure that it did not result from a slow (TEFAP). Samples were amplified for pyrosequencing using forward and growth rate. reverse fusion primers. The forward primer was constructed with the Interestingly, high counts were recorded in salts of Gozo Island Roche A linker (5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3′), an (6.7 log CFU g−1 on DBCM2), Guérande (6.5 log CFU g−1 on CDM) 8–10 bp barcode and the 340F S-D-Arch-0340-a-S-18 primer. The re- and La Palma Flor de Sal (6.4 log CFU g−1 on MGM). Hv-YPC medium verse fusion primer was constructed with a biotin molecule, the Roche supported a lower growth with maximal mean values observed in Blinker(5′-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-3′) and the 915R Gozo Island (4.8 log CFU g−1) and La Palma Flor de Sal (4.7 log CFU g−1). 40 O. Henriet et al. / International Journal of Food Microbiology 191 (2014) 36–44

8

7 MGM CDM 6 Hv-YPC DBCM2

5 salt -1 4

log CFU g 3 * *

* 2 * *

1

0

Fig. 2. Viable counts on four culture media after five weeks of incubation at 37 °C. Each bar represents the sample arithmetic mean on three replicates. The error bar represents the confidence interval for α = 0.05. Stars indicate experiments for which at least one of the replicates gave no colonies at the lowest dilution (undiluted).

This trend was most noticeable in Bolivian, Chardonnay and Pakistani rock The archaeal diversity in Bolivian, Chardonnay, Hâlen Môn, Himalayan salts where no colonies developed on Hv-YPC although they did on the pink, La Baleine coarse sea and Persian blue rock salts was low with iso- other three media. In Camargue and Guérande salts, the difference be- lates related to less than 3 type strains. Most interestingly, all isolates tween the mean count on Hv-YPC and CDM was as high as 3.0 log and from Hâlen Môn salt belonged to genus Halobacterium.Thisgenuswas 2.7 log, respectively. equally important in Himalayan pink salt and La Palma Flor de Sal It is noteworthy that for the three refined salts (Marsel® fine, Jozo® where 13 isolates on MGM medium were related to Halobacterium iodized and uniodized salts), only one viable (corre- noricense, even though the colonies were preferentially selected when sponding to 20 CFU g−1 of salt) was observed in Marsel® fine. The they presented a different phenotype. closest type strain was Staphylococcus hominis subsp. novobiosepticus On the contrary, Guérande and Camargue salts displayed a wide di- GTC 1228T (99.9% identity), which was probably originated from bulk versity. In Guérande salt, isolates were related to 9 type strains from 7 product handling or packaging (data not shown). different genera while in Camargue salt, isolates were related to 9 type strains belonging to 3 genera (Haloarcula, Halobacterium and 3.2. Identification of viable microorganisms Halorubrum). A few extremely halophilic bacteria were also observed, mostly on Colonies isolated on the four hypersaline media were collected and Hv-YPC medium and MGM. These media have a substrate composition the closest type strains were identified by 16S rRNA gene sequencing more complex than CDM and DBCM2, which are supplemented with (Table 2). The thresholds applied to associate an isolate with the pyruvate as sole carbon source. Bacterial isolates related to Salinibacter taxonomic rank of a family, genus or species were 90%, 95% or 97% as ruber (Camargue salt), Halobacillus alkaliphilus and Pontibacillus marinus previously described (Everett et al., 1999; Schloss and Handelsman, (Guérande salts), Actinopolyspora halophila (Gozo Island salt) and 2004; Stackebrandt and Goebel, 1994). The association of two isolates S. hominis (Marsel® fine salt) (data not shown). to Haloferax genus must therefore be taken with great caution since they share only 86.9 and 87.7% of their 16S rRNA gene sequence with the closest Haloferax type strain. Also, 7 isolates shared 90.0 to 95.0% 3.3. Culture-independent assessment of archaeal diversity in food-grade identity with their closest type strain relative and 17 isolates shared salts 95.0 to 97.0%. For these reasons, the presence of genera and in Guérande salt, Haloferax in Chardonnay salt, All culture-based techniques have the important drawback that a Halomicrobium in La Baleine coarse sea salt and Natronomonas in medium with a define composition may not suit the requirement of Camargue salt will not be considered in the following analysis. all species. To overcome this constraint, a metagenomic analysis was The global diversity of viable archaea from the family Halobacteriaceae performed to provide complementary information about the archaeal in food-grade salts was important with 17 genera identified out of the diversity in food-grade salts. A preliminary optimization of the DNA 40 genera validly published to date. The most represented genera extraction step was performed. The most efficient extraction method in- were Haloarcula and Halobacterium with isolates identified in 7 and 12 volved the dissolution of food-grade salt in SW 12%, cell concentration salts, respectively. The second most important genera were Halarchaeum on a 0.2 μm filter, lysis with a solution containing SDS 10% and - (3 salts), Haloplanus (3 salts) and Halorubrum (4 salts). In contrast, many ase K, followed by the DNA purification with phenol/chloroform and genera were identified in only one salt: , Halosarcina and precipitation with isopentanol (data not shown). This method success- Halostagnicola in Gozo Island salt; Halobellus, , fully achieved 16S rRNA gene amplification for eight food-grade salts: La Halolamina and Halopenitus in Guérande salt; Natronoarchaeum in La Baleine coarse, Lac Rose, Palm Island black, Camargue, Dead Sea, Hâlen Palma Flor de Sal; Natrinema in Maras salt. Môn, Maras and Black Sea salts. The PowerSoil® DNA isolation kit O. Henriet et al. / International Journal of Food Microbiology 191 (2014) 36–44 41

Table 2 representing together no more than 0.1% of the population of each salt), Closest type strain of archaeal isolates grown on four culture media. The star indicates that all archaea belonged to the Halobacteriaceae family (Fig. 3). Its members at least two different type strains gave an equal identity percentage. When several isolates are all thriving in hypersaline habitats including crystallizer are related to the same type strain, the range of identity levels is indicated. ponds and salt mines from where many food-grade salts have been ex- For the isolates whose sequences show low similarity with type strains (lower than 95.0 % of identity), the closest non-type sequence in the Ribosomal Database Project Release 11.1 is also given. tracted. Among the 40 genera of this family, 30 have been identified in Closest type strain at least one of the salts and 21 are present in at least 7 of the 9 salts Salt MGM CDM Hv-YPC DBCM2 //closest non-type sequence\\ Identity (%)

T analyzed. 1 Halobacterium jilantaiense NG4 (DQ256409) 98.2 11 1 1Halobacterium noricense A1T (AJ548827) 98.6 to 99.9 The diversity of archaeal genera identified per salt ranges from 21 Black Sea T 1 Haloterrigena longa ABH32 (DQ367242) 97.5 (Hâlen Môn) to 27 (Guérande and Dead Sea). The main genera and 1 Natronomonas moolapensis CSW8.8.11T (AY498645) 98.2 1 Halarchaeum acidiphilum MH1-52-1T (AB371717) 95.6 their relative abundance strongly differ among salts. Four salts display Bolivian 71Halobacterium noricense A1T (AJ548827) 99.8 to 99.9 a single major genus (Palm Island black, Black Sea, Hâlen Môn and 11Haloarcula hispanica ATCC 33960T (AB090167) 98.8 and 99.2 T fi 51 Haloarcula marismortui ATCC 43049 (AY596297) 99.1 to 99.7 Maras) while the other ve have three or more genera with similar oc- 12Haloarcula quadrata 801030/1T (AB010964) 98.0 to 99.2 currence (Fig. 4). Halorubrum is the most abundant genus in Hâlen Môn 1 Haloarcula salaria HST01-2RT (FJ429317) 98.9 11Halobacterium jilantaiense NG4T (DQ256409) 99.5 and 99.7 and Black Sea salts. In the latter, the 16S rRNA gene frequency of Camargue 1 Halorubrum arcis AJ201T (NR028226) 99.3 Halorubrum reaches 84.3%. The gene frequency of Haloarcula accounts 1 Halorubrum lacusprofundi JCM 8891T (NR044766) 99.7 2 Halorubrum lipolyticum 9-3T (DQ355814) 99.6 for 53.9% of the archaeal population in Maras salt and Halonotius is the 1 Halorubrum saccharovorum JCM 8865T (U17364) 98.3 majority genus in Palm Island black salt with a gene frequency of Natronomonas pharaonis DSM 2160T (CR936257) 94.3 1 53.1%. In Lac Rose salt, the archaeal diversity is dominated by three gen- //uncultured archaeon ss004 (AJ969920)\\ 98.6 33 1Halobacterium noricense A1T (AJ548827) 99.3 to 99.7 era: Haloarcula (23.4%), Halorubrum (19.5%) and Natronomonas (16.9%) Haloferax larsenii ZJ206T (AY838278) 86.9 1 whereas in Dead Sea salt, 79.4% of the population is made up by 4 Chardonnay //uncultured archaeon EV818CFSSAHH131 (DQ336966)\\ 96.1 Haloferax sp.* (AY838278 and DQ860977) 87.7 genera: Halorubrum (26.5%), Halomicrobium (23.2%), Halorhabdus 1 //uncultured archaeon EV818CFSSAHH131 (DQ336966)\\ 96.4 (18.1%) and Halobacterium (11.5%). 12 Haloarcula japonica JCM 7785T (EF645685) 98.0 to 99.7 1 Halobacterium jilantaiense NG4T (DQ256409) 98.1 1 A1T (AJ548827) 98.1 Haloplanus aerogenes TBN37T (GQ282625) 94.2 and 96.3 11 Gozo Island //Halobacteriaceae archaeon GX21 (HM063950)\\ 98.1 1 Halorhabdus tiamatea SARL4BT (EF127229) 95.9 1 Halorubrum xinjiangense BD-1T (AY510707) 99.4 Genus Food grade salt T 1 Halosarcina pallida BZ256 (EF055454) 99.5 Lb Pi Hm Gu Ca Ma Lr Ds Bs 1 Halostagnicola larsenii XH-48T (AM117571) 99.7 T Halorubrum 1 Halobellus inordinatus YC20 (JQ237122) 99.3 5 Halogeometricum rufum RO1-4T (EU887286) 99.6 to 100.0 Halobacterium 4 Halolamina pelagica TBN21T (GU208826) 99.2 to 99.5 Haloarcula Halomicrobium mukohataei JCM 9738T (EF645690) 92.0 and 92.4 Halonotius 2 //Halobacteriaceae archaeon YC89 (JQ237120)\\ 97.6 Halorhabdus //uncultured Halorhabdus sp. SFH1C101 (FN391257)\\ 99.3 Haloquadratum 1 Halopenitus persicus DC30T (JF979130) 95.3 Guérande 1 Halorubrum sp.* (AM900832 and U17364 ) 99.7 Halomicrobium 1 Halosimplex carlsbadense 2-9-1T (AJ586107) 95.9 Natronomonas 1 Haloterrigena jeotgali A29T (EF077633) 98.4 Haloplanus 2 Haloterrigena longa ABH32T (DQ367242) 96.2 and 97.0 Halopelagius T 1 DSM 5511 (CP001860) 95.1 Halosimplex Natronomonas moolapensis CSW8.8.11T (AY498645) 93.9 1 Haloferax //uncultured archaeon ss004 (AJ969920)\\ 97.8 Hâlen Môn 1 1 1 3 Halobacterium noricense A1T (AJ548827) 99.5 to 99.9 T Himalayan 2 Halarchaeum acidiphilum MH1-52-1 (AB371717) 97.8 and 97.9 Natronococcus pink 13 1 2 Halobacterium noricense A1T (AJ548827) 96.6 to 99.9 Haladaptatus 1 Haloarcula marismortui ATCC 43049T (AY596297) 98.7 La Baleine Halomicrobium mukohataei JCM 9738T (EF645690) 93.6 coarse sea 1 Halogeometricum //uncultured haloarchaeon XKL4 (JN714407)\\ 99.7 T Halococcus 11 Haloarcula marismortui ATCC 43049 (AY596297) 93.6 and 99.8 13 Halobacterium noricense A1T (AJ548827) 97.4 to 99.8 Halogranum 1 Haloplanus aerogenes TBN37T (GQ282625) 99.0 Haloterrigena T La Palma 3 Haloplanus vescus JCM 16055 (EU931578) 96.6 to 98.4 Natrinema Flor de Sal 1 Halorubrum sp.* (U17364 and DQ355814) 98.4 T 1 Halorubrum tibetense 8W8 (AY149598) 96.6 Natronoarchaeum 1 Halosimplex rubrum R-27T (HM159603) 99.1 T 1 Natronoarchaeum mannanilyticum YSM-123 (AB501360) 97.5 1 Haloarcula hispanica ATCC 33960T (AB090167) 99.3 Halarchaeum Lac Rose 11Halobacterium noricense A1T (AJ548827) 99.4 and 99.4 Halopiger 2 Natronomonas gomsonensis SA3T (JF950943) 95.6 and 98.8 Halomarina T 11Haloarcula hispanica ATCC 33960 (AB090167) 98.2 and 98.4 Halosarcina T 97.9 to 99.4 26 1Haloarcula marismortui ATCC 43049 (AY596297) 1 Haloarcula vallismortis CGMCC 1.2048T (EF645688) 99.2 1 Halobacterium noricense A1T (AJ548827) 99.7 Archaeoglobus Maras 2 Haloplanus vescus JCM 16055T (EU931578) 96.1 and 97.6 Thermococcus 1 Halosimplex carlsbadense 2-9-1T (AB072815) 98.7 Palaeococcus 1 Natrinema versiforme XF10T (AB023426) 98.2 Methanosaeta 1 Natronomonas pharaonis DSM 2160T (CR936257) 95.7 T Genera identified 25 23 21 27 22 22 22 27 26 11Haloarcula marismortui ATCC 43049 (AY596297) 96.6 and 99.0 T Pakistani 1 Haloarcula salaria HST01-2R (FJ429317) 99.6 25 < x ≤ 100 rock T 1 Haloarcula vallismortis CGMCC 1.2048 (EF645688) 97.5 10 < x ≤ 25 31 Halobacterium noricense A1T (AJ548827) 98.4 to 99.7 T 5 < x ≤ 10 Persian blue 1 Halarchaeum acidiphilum MH1-52-1 (AB371717) 97.9 T ≤ rock 11 3Halobacterium noricense A1 (AJ548827) 97.7 to 99.9 1 < x 5 0.5 < x ≤ 1 0 < x ≤ 0.5 No occurrence (Mo-Bio) was required for Guérande salt because of its humic acid content. Fig. 3. Relative abundance of archaeal genera in nine food-grade salts from worldwide origin High-throughput sequencing on 16S rRNA gene from archaeal cells in as determined by high-throughput sequencing. The color code indicates the range of relative 9 food-grade salts from diverse origins revealed that, with only few ex- abundance for a given genus. Lb, La Baleine coarse sea; Pi, Palm Island black; Hm, Hâlen Môn; ceptions (Palaeococcus, Archaeoglobus, Thermococcus and Methanosaeta, Gu, Guérande; Ca, Camargue; Ma, Maras; Lr, Lac Rose; Ds, Dead Sea; Bs, Black Sea. 42 O. Henriet et al. / International Journal of Food Microbiology 191 (2014) 36–44

Gu Lb Lr Haladaptatus Haloarcula Halobacterium Halobaculum Halococcus Haloferax Halogeometricum

Pi Ca Ds Halogranum Halomicrobium Halonotius Halopelagius Haloplanus Haloquadratum Halorhabdus Halorubrum Hm Ma Bs Halosimplex Haloterrigena Natrinema Natronobacterium Natronococcus Natronomonas Other genera

Fig. 4. Relative abundance of archaeal genera in salts, accounting individually for more than 0.5% of the whole archaeal population as determined by high-throughput sequencing. Gu, Guérande; Lb, La Baleine coarse sea; Lr, Lac Rose; Pi, Palm Island black; Ca, Camargue; Ds, Dead Sea; Hm, Hâlen Môn; Ma, Maras; Bs, Black Sea.

Finally, Haloquadratum, the most commonly found genus in solar The reported to survive in inclusions correspond salterns, is the second genus in order of importance in La Baleine to the isolates most often observed in our study. This supports the (23.8%) and Camargue salts (15.6%) although in 4 salts (Hâlen Môn, hypothesis that the diversity in food-grade salts would be oriented by Guérande, Maras and Dead Sea salts), its relative occurrence is lower the survival abilities in crystal inclusions. than 1%. 4.2. Congruence and discordance between metagenomic and culture-based methods 4. Discussion Comparison of the archaeal genera identified by metagenomics with 4.1. From waters to food-grade salts: a matter of survival? the isolates grown on four culture media revealed congruence for the Maras, Lac Rose and Camargue salts but discordance for the Black Sea, Metagenomic analysis was performed on 9 food-grade salts produced Guérande, La Baleine coarse sea and Hâlen Môn salts. by solar evaporation of hypersaline waters. These extreme environments In Maras salt, isolates closely related to Haloarcula, Halobacterium, were reported to maintain high densities of halophilic archaea dominated Haloplanus, Halosimplex and Natronomonas were identified in agree- by a limited number of genera, mostly Haloquadratum, Halorubrum and ment with the metagenomic analysis where these genera represented Haloarcula (Bidle et al., 2005; Bowman et al., 2000; Oh et al., 2010; altogether 89.3% of the archaeal population. The isolates from Lac Rose Pašić et al., 2005). In our study, haloarchaeal diversity in food-grade salt were related to Haloarcula, Halobacterium and Natronomonas, salts revealed that Halorubrum, Haloarcula or Halobacterium was domi- three main genera from the metagenomic diversity. In Camargue salt, nant in at least one salt. On the contrary, Haloquadratum appeared to be isolates were related to Haloarcula, Halorubrum, Halobacterium and a minor actor of food-grade salt diversity. This is interesting since this Natronomonas, representing 53.5% of the population. genus has been repeatedly reported as a major in solar saltern On the contrary, isolates from Black Sea salt were related to waters (Burns et al., 2004; Oh et al., 2010). Halobacterium, Haloterrigena and Natronomonas although metagenomic Similar conclusions arose from a comparison of the haloarchaeal diversity indicated a large majority of Halorubrum members. In diversity reported in Maras saltern waters (Maturrano et al., 2006) Guerande salt, none of the three most represented genera by with the corresponding salt analyzed in the present study. The anal- metagenomics (Halobacterium, Halorhabdus and Haloplanus)wereiden- ysis of a 16S rRNA gene clone library generated from saltern waters tified on culture media. In La Baleine coarse sea and Hâlen Môn salts, the indicated that 69% of the archaeal clones were closely related to genera isolated were minor actors of the corresponding metagenomic di- Hqr.walsbyi and the rest to . However, our versity. Besides, two salts used in the pyrosequencing experiment did study identified 3 major genera in Maras salt (Haloarcula, Halobacterium not give rise to colony growth: Palm Island black and Dead Sea salts. and Natronomonas)butHqr. walsbyi was not detected. The Haloquadratum and Halonotius genera were not isolated on the In the process of salt crystallization, most haloarchaea present in the four media selected in this study although they were among the main salterns are trapped inside fluid inclusions (Norton and Grant, 1988). genera in Palm Island black, La Baleine coarse sea or Camargue salts. Most of the strains isolated from salt inclusions were related to The medium proposed to grow Hqr. walsbyi and Halonotius strains is Halorubrum, Halobacterium, Haloarcula and Halococcus (McGenity DBCM2 (Burns et al., 2010; Dyall-Smith, 2009). This medium was in- et al., 2000; Mormile et al., 2003). Long-term survival of isolates related cluded in this study, with the notable differences that vitamin solution to Halorubrum, Natronomonas and Haloterrigena inside salt crystals was and MGM 23% at 50 ml/l were omitted. It is unlikely that the absence suggested to exceed 22,000 years (Schubert et al., 2010). of vitamin solution could cause a growth inhibition, as the medium O. Henriet et al. / International Journal of Food Microbiology 191 (2014) 36–44 43 used by another research group did not contain vitamin complements this article can be found, in the online version, at http://dx.doi.org/10. (Bolhuis et al., 2004). On the contrary, the addition of a very low concen- 1016/j.ijfoodmicro.2014.08.019. tration of yeast extract either directly in the preparation or through MGM 23% supplementation may have an impact on the growth of Conflict of interest these archaea. It must also be noted that the recovery of Haloquadratum from natural samples is highly improved in liquid medium (Bolhuis The authors declare no conflict of interest. et al., 2004). ThreegeneraisolatedinGuérandesaltwerenotfoundinthecorre- Acknowledgments sponding metagenomic analysis: Halobellus, Halolamina and Halopenitus. As the 16S rRNA gene sequences of their type strains is covered by the We are deeply indebted to Antoine Toussaint and François Louesse primers used in the analysis, it seems most likely that their absence is for their contribution in the early stage of this research and to Annika caused by either a low occurrence in the archaeal community or a poor Gillis and Thomas Vanzieleghem for their critical reading of the manu- efficiency in the extraction of their DNA, possibly caused by some pecu- script and their helpful comments. We are also grateful to John Delton liarities in their cellular structure. Hanson from the Research and Testing Laboratory for helpful sugges- tions and comments on the metagenomics methodology. This work was supported by the Université catholique de Louvain. 4.3. Exploring the slow-growing haloarchaeal diversity among fast- growing halophilic bacteria References

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