Annals of Microbiology (2018) 68:851–861 https://doi.org/10.1007/s13213-018-1391-6

ORIGINAL ARTICLE

Isolation and culturing of protease- and lipase-producing agarilyticus GUGFAWS-3 from marine Haliclona sp. inhabiting the rocky intertidal region of Anjuna in Goa, India

Sanket Krishnanath Gaonkar1 & Irene Jeronimo Furtado1

Received: 23 March 2018 /Accepted: 24 October 2018 /Published online: 19 November 2018 # Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan 2018

Abstract Three extremely halophilic bionts GUGFAWS-1, GUGFAWS-2, and GUGFAWS-3 were isolated from a marine, white sponge, attached to rocks, in the intertidal region of Anjuna, Goa, India (15° 34′ 05″ N, 73° 44′ 17° 40′ E). Because the sponge had irregular tubes arranged in clusters, it was identified as Haliclona sp. All sponge bionts produced protease and lipase. GUGFAWS-1 and GUGFAWS-2 were euryhaline Eubacteria, growing from 0 to 30% NaCl concentration. The biont, GUGFAWS-3, was a haloarchaeon having glycerol diether moieties in its cells and showed R-O-R and long isoprenoid chains, in FTIR. The haloarchaeon, GUGFAWS-3, simultaneously produced extracellular 49.5 U mL−1 of protease and 3.67 U mL−1 of lipase, in the presence of 25% NaCl. It grew as dark orange-red colonies at 5–30% NaCl. Its growth was sensitive to bile salts and resistant to 700 U of penicillin. Cells were Gram-negative cocci, arranged in pairs, and 1-μm size in SEM micrograph. It possessed bacterioruberin with absorption at 387, 468, 492, and 523 nm. The 16S rRNA gene sequence of GUGFAWS-3 was 99.1% similar to Halococcus agarilyticus 62E (T) of the family Halococcaceae of the domain . This study is the first evidence of retrieval and culturing of Halococcus agarilyticus strain GUGFAWS-3 (MF425611) from marine Haliclona sp. with ability to simultaneously produce protease and lipase extremozymes of ecological and biotechnological significance.

Keywords Marine Haliclona sp. . . Protease . Lipase . Extremozymes

Introduction or fatty acid ester. Proteases (3.4.21-24) hydrolyze proteina- ceous material of plant and animal origin (Zhao et al. 2008). Globally, haloarchaea and extremely halophilic eubacteria are Extracellular lipases with optimum activity in the range of investigated for the production of commercially important 3–4 M NaCl have been isolated from Natronococcus sp. proteases and lipases, which function at 3–4 M NaCl and (Bhatnagar et al. 2005; Boutaiba et al. 2006), Haloarcula water activity of aw 0.75 (Grant 2004; Gupta et al. 2015). marismortui (Camacho et al. 2009), and from unidentified Lipases (3.1.1.3) and esterases (3.1.1.1) hydrolyze long- haloarchaeal strains (Ozcan et al. 2009). To this day, only a chain fatty acyl-glycerol and short-chain acyl-glycerol, respec- single lipase from Haloarcula sp. G41 has been purified and tively. These enzymes carry out amidation, transesterification, studied for biotechnological application by Xin and Hui-Ying and enantioselective reactions (Matsumoto et al. 2004). Their (2014). Similarly, proteolytic enzymes with optimal activity in production is induced by oil, fatty acid, fatty acid alcohol, and/ the range of 3–4.3 M NaCl have been reported by: Norberg and Hofsten (1969)inHalobacterium salinarium strain I, Izotova et al. (1983)inHalobacterium halobium,Kamekura and Seno (1993)inHaloferax mediterranei R4, Vidyasagar Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13213-018-1391-6) contains supplementary et al. (2006)inHalogeometricum borinquense strain TSS101, material, which is available to authorized users. Shi et al. (2006)inNatrinema sp. R6-5, Manikandan et al. (2009)inHaloferax lucentensis VKMM007, and Akolkar * Irene Jeronimo Furtado et al. (2009)inHalobacterium sp. SP1. [email protected] As intertidal reaches are exposed to tidal fluxes with fluc- tuating salinity and also to effluents, having animal and petro 1 Department of Microbiology, Goa University, Taleigao Plateau, Goa 403 206, India industries waste, we envisaged that sponges from these 852 Ann Microbiol (2018) 68:851–861 reaches would be a good source of extremely halophilic bionts were incubated at RT for 15 days, and checked for growth with polymer-degrading capabilities. Earlier, Velho-Pereira or inhibition of growth. and Furtado (2014) reported haloarchaeal bionts from marine The resistance of each isolate to antibiotics was evaluated sponges, inhabiting Mandapam, India. by spread plating, individual cultures, in their log phase, on During this study, we successfully isolated and character- modified Miller-Hinton agar with 25% NaCl (w/v), for lawn ized a biont GUGFAWS-3, as Haloccoccus sp., and the host growth. Antibiotic Discs (octa-disks; HiMedia Laboratories sponge as Haliclona sp. Interestingly, this biont simultaneous- Ltd., Mumbai, India) impregnated with appropriate antibiotic ly produces protease and lipase extremozymes, functional in such as bacitracin (5 μg), penicillin (10 U), ampicillin (5 μg), the presence of 25% NaCl, and is of great commercial and ciprofloxacin (5 μg), streptomycin (10 μg), vancomycin biotechnological significance. (30 μg), gentamycin (10 μg), and linezolid (30 μg) were placed on agar and monitored after 15 days for zone of inhi- bition of growth. Materials and methods Phenotypic characterization of microbial bionts Isolation of microbial bionts from the marine sponge The three distinct isolates GUGFAWS-1, GUGFAWS-2, and GUGFAWS-3 were individually subjected to Gram reaction. A piece of 2 × 2 cm2 was excised from a marine sponge, Phenotypic characteristics of cell morphology, catalase, nitrate growing attached to rocks in the intertidal region (located at reductase, and motility were examined as described by 15° 34′ 05″ N, 73° 44′ 17° 40′ E), in Anjuna, Goa, India. The Smibert and Krieg (1994). sponge sample was washed and macerated, using sterile motor Cellular morphology of each of the three halophilic isolates and pestle. The macerate was added to 100 mL of sterile NaCl GUGFAWS-1, GUGFAWS-2, and GUGFAWS-3 was deter- Tryptone Yeast Extract Medium (NTYE) (Braganza and mined, using a light microscope (Nikon H600L) and 100X oil Furtado 2009) consisting of 20 g MgSO , 5 g KCl, 0.2 g 4 immersion lens. Gram character was studied using the modi- CaCl , 3 g yeast extract, 5 g tryptone, and 250 g crude salt/ 2 fied gram staining method of Dussault (1955). The slide was NaCl per liter, pH 7 (adjusted with 1 N NaOH). Penicillin to air dried and observed under oil immersion lens. final concentration of 300 U/mL was also added. Flasks were Biochemical tests such as utilization of citrate and produc- incubated in a REMI BOD CIS-24 PLUS at 150 rpm, at 30 °C. tion of acid from D-glucose, sucrose, and lactose were done After 8 days, an aliquot of this growing culture broth was according to Norberg and Hofsten (1969). Stocks of D-glu- serially diluted and spread on to NTYE agar. Plates were in- cose, sucrose, and lactose were prepared separately and added cubated at room temperature (RT, 28 to 30 °C) for 15 days. to the basal medium to a final concentration of 0.5% (v/v). Three distinct colonies were picked, purified, and designated Each tube was inoculated with 2% (v/v) inoculum of each of as GUGFAWS-1, GUGFAWS-2, and GUGFAWS-3 denoting the three isolates separately in 20% NaCl with OD of 1 at GU (Goa University), G (Gaonkar), F (Furtado), A (Anjuna), A . All tubes were incubated at RT for 8 days. WS (white sponge), and 1, 2, and 3 as strain numbers. 600 Thin smear of cells of GUGFAWS-3 freshly grown in a synthetic medium with 20% NaCl (NSM) (Aguiar and Physiological characterization of microbial bionts Furtado 1996; Raghavan and Furtado 2004) was prepared on a clean glass coverslip (2 × 2 cm), air dried, and exposed Isolates GUGFAWS-1, GUGFAWS-2, and GUGFAWS-3 to 2.0% (v/v) glutaraldehyde in 20% NSM at RT overnight. were grown separately in (i) Tryptone Yeast extract (TYE) Coverslip was exposed to an increasing gradient of acetone- broth (Aguiar and Furtado 1996)consistingof(g/L)20 water of 30%, 50%, 70%, and 90% each for 10 min, respec-

MgSO4, 5 KCl, 0.2 CaCl2, 3 yeast extract, and 5 tryptone, tively, and finally to 100% acetone. Coverslip was air dried pH 7 and (ii) TYE with salt concentration ranging from 5 to and mounted on to an SPI-Module sputter and observed under 30% at RT, pH 7, on a rotary shaker at 150 rpm. Growth was SEM (ZEISS E VO 18 special edition) for morphological measured at 600 nm using a Shimadzu UV–Vis details. spectrophotometer-1601 (Japan). The isolates were spot inoculated on the NTYE agar medi- Preparation of whole cells and chemotaxonomic um with pH varying from 5, 6, 7, 8, and 9 and incubated at RT analysis for 15 days. The temperature required for optimum growth of the isolates was determined, by inoculating culture on NTYE GUGFAWS-1, GUGFAWS-2, GUGFAWS-3 were individu- agar and incubating at RT, 37 °C and 45 °C. ally grown in NTYE at RT and 150 rpm. After 8 days, cells Individual cultures were spot inoculated onto were harvested at 2432g for 10 min at 4 °C using an MacConkey’s agar containing 25% NaCl (w/v) and plates Eppendorf 5417 R centrifuge. Cells of each culture were Ann Microbiol (2018) 68:851–861 853 resuspended separately to an absorbance 2 at 600 nm and Culturing of Halococcus sp. for production studied for their susceptibility to water (Fukushima et al. of extracellular protease and lipase 2007), pigment profile (Raghavan and Furtado 2005), and presence of glycerol diether moieties (GDEMs) (Ross et al. Isolate, GUGFAWS-3, pre-grown in the NSM liquid medi- 1981). Haloferax alexandrinus KF796625 was used as a stan- um with 0.2% glucose (NGSM), was inoculated into the dard haloarchaeon. NSM liquid medium with 0.5% (w/v) gelatin/0.5% (v/v) tween 80. Flasks were incubated at RT at 150 rpm. The Fourier transform infrared spectroscopy medium with appropriate substrate and without GUGFAWS-3 was used as a control. Aliquots of growing culture were used to monitor growth and production of pro- The spot resolved at Rf of 0.2 was scrapped from TLC plate and extracted in hexane, dried, and mixed with KBr powder in tease and lipase every 24 h. 1:10 (w/w). The KBr pellet was analyzed by Fourier transform infrared spectroscopy (FTIR) (IRPrestige-21 Shimadzu) in the Evaluation of concomitant production of protease range of 4000–400 cm−1 at a resolution of 4 cm−1. and lipase by Halococcus sp. MF425611

The culture pre-grown in NTYE was inoculated in NTYE Statistical analysis with 0.2% tween 80 incubated at RT at 150 rpm. Growth and simultaneous production of protease and lipase were de- The key phenotypic characters for each isolate were assessed termined every 24 h. and rated as 0 (for negative) or 1 (for positive), respectively. The similarity matrix was calculated by dividing the size of Enzyme assays the intersection of two attributes by the size of their union, using the Jaccard coefficient. Percentage similarity between The culture broth was centrifuged at 9217g at 4 °C for 10 min. the bionts was analyzed by cluster analysis based on a paired The clear cell-free supernatant (CFS) was used as crude en- grouped algorithm. The computation was done using Past ver. zyme sample. 2.17c (Hammer et al. 2001) and the phenogram was construct- ed, thereof, to derive the phenotypic relationship between Protease assay GUGFAWS-1, GUGFAWS-2, and GUGFAWS-3. A standard Kunitz assay (1947) was employed with minor Screening of bionts for production of extracellular modification. Briefly, 0.2 mL of crude enzyme was incubated enzymes with 0.8 mL of 0.5% (w/v) casein in 0.1 M Tris HCl buffer pH 7 containing 2 M NaCl. The mixture was incubated at Detection of production of 15 different extracellular hydrolyt- 37 °C. After 15 min, 0.5 mL of 10% (w/v) trichloroacetic acid ic enzymes was performed on 20% NSM agar plates in tripli- (TCA) was added to stop the reaction. After 20 min, the reac- −1 cates. The medium consists of (gL )20MgSO4,13MgCl2,1 tion mixture was centrifuged (10,000g, 4 °C, 10 min) and CaCl2,4KCl,2NH4Cl, 0.2 NaHCO3,0.005FeCl3,0.5 protease activity of supernatant was checked by monitoring KH2PO4, and 200 NaCl, pH of media was adjusted to 7 with formation of tyrosine. A control consisting of only substrate 1 N KOH. Substrates for protease/amylase/cellulase/ and buffer was also maintained. One unit of protease activity pectinase/DNase/chitinase/tannase/esterase/urease/agarase was defined as the amount of enzyme required to liberate 1 μg and for lipase/glutaminase/inulinase/xylanase and phospha- of tyrosine per min. tase to a final concentration of 5 gL−1,2gL−1,and5mM, respectively, were added to the NSM medium. Isolates Lipase assay GUGFAWS-1, GUGFAWS-2, and GUGFAWS-3 were spot inoculated on to substrate NSM agar plates, incubated for Lipolytic activity was estimated according to the modified growth at RT for 15 days. For the detection of cellulase, method of Beisson et al. (2000) wherein para-nitrophenyl ac- xylanase, and chitinase, the plates were flooded with 0.1% etate was used as a substrate. A 10 mM substrate solution was (w/v) Congo red dye for 30 min, followed by washing twice prepared in isopropanol and 1 mL aliquot was mixed with with 1 M NaCl for 5 min each. Agarase, esterase, inulinase, 9 mL of a solution containing 100 mM Tris buffer at pH 7, pectinase, amylase; DNase, and protease were visualized by 0.5% (v/v) Triton X-100, and 2 M NaCl and warmed in a water

Lugol’s iodine, 1 N HCl, and 15% (w/v)acidicHgCl2,respec- bath at 30 °C. The reaction mixture consisted of 0.8 mL of tively. Formation of the zone of clearance/colored halos/pre- substrate emulsion and 0.2 mL of crude enzyme supernatant cipitation around the colony was recorded. Only in the case of was incubated for 10 min and the amount of para-nitrophenol lipase, a clear zone or precipitate zones were formed directly. released was estimated at 410 nm against blank. One enzyme 854 Ann Microbiol (2018) 68:851–861 unit (U) was referred as the amount of enzyme liberating 1 μM Results and discussion of p-nitrophenol per minute under assay condition. The molar extinction coefficient for p-nitrophenol was calculated under Isolation and culturing of extremely halophilic bionts same conditions as the assay and used to calculate the enzyme from marine Haliclona sp. activity. The intertidal region, located at 15° 34′ 05″ N, 73° 44′ 1740′ E in Anjuna, Goa, is rocky and is subjected to tidal changes and Stability of crude lipase produced along with protease environmental stresses. Waters of the region are turbid and had particulate matter and an oily film on their surface. The crude enzyme stored at 4 °C was verified for its stability, Several invertebrates and sponges are reported from this hab- everyday over a period of 5 days. Lipase activity on day 1 was itat (Dahihande and Thakur 2017). Since sponges are filter referred to as t0 (t0 = initial lipase activity). feeders, we envisaged that waters carrying oily material, par- ticulate matter, and microbes would reach the choanocytes during influx, and possibly attach to cell surfaces and establish Molecular characterization of GUGFAWS-3 therein as bionts. Since the sponge under experiment grew on rocks as A single colony of GUGFAWS-3 was grown in the NTYE creamish white irregular tubes, in clusters, as seen in Fig. 1a, medium for 8 days. Cells were harvested by centrifuging at it was identified as belonging to Haliclona sp. of the family 9727g at 4 °C for 10 min. Genomic DNA was obtained by Haliclonidae,orderHaplosclerida,classDemospongiae,and suspending the cell in distilled water and boiling for 10 min at phylum Porifera as per Grant (1841). Macerate of this sponge 60 °C, cooling at RT and centrifuged at 9217g at 4 °C for in NTYE with 300 U of penicillin resulted in deep creamish 10 min. The gene for 16S rRNA was amplified with primers homogenous culture within 8 days indicating that the sponge A109 (F) AC(G/T)GCTCAGTAACACGT and 1510 (R) tissue harbored extremely halophilic microbial bionts able to GGTTACCTTGTTACGACTT. The Eppendorf thermocycler grow in 25% NaCl. Three distinct bionts namely GUGFAWS- was programmed for DNA denaturation initially at 95 °C for 1, GUGFAWS-2, and GUGFAWS-3 were isolated. Such ex- 5min,followedbydenaturationat95°Cfor1min,primer tremely halophilic bionts were isolated earlier for the first time annealing at 53.5 °C for 1 min, strand extension at 72 °C for from sponges inhabiting Mandapam in India by Velho-Pereira 1 min (35 cycles), and a final elongation step at 72 °C for and Furtado in 2014. 10 min. The amplified DNA was purified using PCR purifi- cation kit (GeneJET PCR Purification Kit, Thermo Fisher Scientific). The 16S rRNA amplicon corresponding to ap- The morphological, physiological, and biochemical proximately 1.5 Kb were bidirectionally sequenced using the characteristics of sponge bionts Big Dye terminator sequence method (BigDye® Terminator v3.1 Cycle Sequencing Kit, Applied Biosystems, India). The The key characteristics of GUGFAWS-1, GUGFAWS-2, and reactions were analyzed through an automated DNA sequenc- GUGFAWS-3 are detailed in Table 1.Asseeninonlinere- er (Applied Biosystems) and electrograms reported in source 1, bionts GUGFAWS-1 and GUGFAWS-2 grew be- ChromasPro version 2.6.4. tween 0 and 30% NaCl and with growth optimum at 5 and Sequences obtained were manually curated, checked for 10% NaCl, respectively. They were termed as euryhaline bac- query coverage using BLASTn, and uploaded in the teria according to terminology by Ventosa et al. (1998); Ribosomal Database Project (RDP release 11) for alignment GUGFAWS-3, which grows between 5 and 30% NaCl with using the Hierarchy browser (Cole et al. 2014). optimum growth at 20% NaCl, was termed as truly extremely The possible generic or level identity was halophilic as per guidelines of Grant et al. (2001). The bionts worked out, based on similarity/dissimilarity in 16S rRNA GUGFAWS-2 and GUGFAWS-3 grew at a pH range of 5–9, homology and phenotypic characteristics. Reliability of while GUGFAWS-1 grew in a pH range of 7–9. The growth of taxonomic identity to species level was arrived in case of all bionts was the maximum at RT (Table 1). > 97% 16S rRNA sequence homology to a single describ- On NTYE, the bionts GUGFAWS-1 and GUGFAWS-2 able type strain in both RDP-II and EZ taxon-e database. formed circular, opaque, smooth, butyrous, and cream colo- The taxonomic identity of the 16S rRNA gene sequence of nies. The GUGFAWS-3 formed dark orange-red colonies as strains and its percentage similarity with other taxa was seen in Fig. 2a (i). Cells of GUGFAWS-1 and GUGFAWS-2 calculated using the BIdentify^ option of the EzTaxon-e were Gram-negative and short rods. Cells of GUGFAWS-3 server (Kim et al. 2012). The 16S rRNA gene sequence were Gram-negative cocci arranged in pairs as seen in was deposited in NCBI GenBank with accession number Fig. 2a(ii)and1-μm size in SEM micrograph as recorded in MF425611. Fig. 2a(iii). Ann Microbiol (2018) 68:851–861 855

Fig. 1 GDEM profile of extremely halophilic bionts isolated from Haliclona sp. a Marine Haliclona sp. b TLC of: lane 1, GUGFAWS-1; lane 2, GUGFAWS-2; lane 3, GUGFAWS-3; and lane 4, Haloferax alexandrinus KF796625 of glycerol diether moieties (GDEMs); chromato- graph developed in the ether, diethyl ether (85:35 v/v), and vi- sualized with 10% (w/v)ethanolic phosphomolybdic acid reagent

GUGFAWS-1 and GUGFAWS-2 were resistant to sodium The FTIR of this spot showed the presence of ether C–O–C taurocholate while GUGFAWS-3 was found to be sensitive. groups with absorption in the range of 1070–1200 cm−1 GUGFAWS-1 was sensitive to all the antibiotics tested. (Fig. 2c), which confirmed the presence of GDEM, a charac- GUGFAWS-2 was resistant to bacitracin, penicillin, ampicil- teristic feature of members of phylum Euryarcheota of do- lin, streptomycin, vancomycin, and gentamycin but sensitive main Archaea. to ciprofloxacin and linezolid. GUGFAWS-3 was resistant to penicillin, bacitracin, and ampicillin. All the three sponge Statistical analysis bionts fermented glucose were methyl red and Voges- Proskauer negative and catalase positive. Bionts PAST analysis sorted the three sponge bionts based on their GUGFAWS-2 and GUGFAWS-3 showed nitrate reductase. key differentiating phenotypic characteristics (Table 1), using Only GUGFAWS-1 was non-motile. similarity matrix and paired group algorithm cluster analysis, Cells of GUGFAWS-3 lysed in distilled water, whereas the as depicted in the phenogram (online resource 2). Thus, PAST other two bionts were resistant to lysis in distilled water. analysis confirmed their biochemical differences and identifi- Unlike Halococcus (Fukushima et al. 2007) which lysed in cation up to genera which have been sorted according to keys 10 days, GUGFAWS-3 lysed in 15-min exposure to water. of Bergey’s Manual of Systematic Bacteriology (Sneath et al. GUGFAWS-3 was orange-reddish in color. As seen in 1986). These key differential characteristics of GUGFAWS-1 Fig. 2b, chloroform extracted the negligible amount of pig- and GUGFAWS-2 indicated their affiliation to genera ment from GUGFAWS-3 cells. Substantial pigment was Acinetobacter sp. and Chromohalobacter sp., respectively, extracted in acetone:methanol (1:1 v/ v )orin of the domain Eubacteria and GUGFAWS-3 to Halococcus chloroform:methanol (1:1 v/v) or in acetone. All three sp. of the domain Archaea. extracts gave identical profiles in the visible range but differed in the pigment intensity. Extractability by metha- Extremely halophilic enzyme potential of bionts nol is closely comparable with that by acetone:chloroform (1:1 v/v). Pigment extract with all of these solvents GUGFAWS-1, GUGFAWS-2, and GUGFAWS-3 were showed an absorption maximum at 523, 492, 468, and checked for production of extracellular hydrolytic enzymes, 387 nm. These absorption maxima correspond to the spec- as these bionts were isolated from sponge habitat waters hav- tral features of 50-carbon open-chain carotenoid, ing dissolved organic matter (DOC) containing proteins, lipids bacterioruberin, and its derivatives with absorption maxi- of microbial, and plant and animal origin (Basu et al. 2011). ma at 467, 493, and 527 nm, for three-fingered peaks and Keeping with this and as seen in Fig. 3, all the three bionts at 370 and 385 nm, for two cis peaks (Raghavan and produce protease, lipase, and esterase. The maximum produc- Furtado 2005). tion of these enzymes was by GUGFAWS-3 with zone index According to Purdy et al. (2004), presence of GDEM is the of protease > lipase > esterase. The culture also produces principal key for assigning affiliation of domains to isolates. amylase, pectinase, inulinase, chitinase, tannase, and agarase. Only the hexane extracts of cells of GUGFAWS-3 revealed a None of the isolates showed cellulase, xylanase, DNase, ure- single spot of Rf at 0.2 corresponding Rf value to the spot of ase, and phosphatase activities. Only GUGFAWS-2 produced GDEM of Haloferax alexandrinus KF796625 (Fig. 1b). glutaminase. 856 Ann Microbiol (2018) 68:851–861

Table 1 Differential characteristics between sponge Characteristics 1 2 3 4 bionts and Halococcus agarilyticus strain 62ET Shape Short rods Short rods Cocci Cocci Gram character –– –– Motility Non - motile Motile Motile Non- motile NaCl range (%) 0–30 0–30 5–30 15–30 NaCl optimum (%) 5 10 20 20–30 pH range 7–95–95–96.5–8.5 pH optimum 7 7 9 7.5 Temperature range (°C) RT-37 RT-37 RT-45 22–47 Temperature optimum RT RT RT 42 (°C) Bacitracin S R R S Penicillin G S R R R Ampicillin S R R R Ciproflaxin S S S ND Streptomycin S R S R Vancomycin S R S ND Gentamycin S R S R Linezolide S S S R Na-taurocholate R R S ND Pigmentation Cream Cream Orange Pink Carotenoid Nil Nil 468, 492, 523 ND GDEM Nil Nil + + Lysis with D/W Nil Nil + Nil Glucose + + + Nil Sucrose + + Nil + Lactose Nil + + Nil Indole Nil Nil + W Methyl red Nil Nil Nil ND Voges pros Nil Nil Nil ND Citrate Nil + Nil Nil Nitrate Nil + + + Catalase + + + W Domain Eubacteria Eubacteria Archaea Archaea Aceinatobacter Chromohalobacter Halococcus Halococcus sp. sp. sp. sp.

Strains 1, GUGFAWS-1; 2, GUGFAWS-2; 3, GUGFAWS-3; and 4, Halococcus agarilyticus strain 62ET (data taken from Minegishi et al. (2015). +, present; Nil, absent; −, negative; W, weak positive; ND, not determined; RT, 28 to 30 °C; GDEM, glycerol diether moieties; R,resistance;S,sensitive

Production of protease and lipase by biont by 50% with 0.5% gelatin and by 75% with 0.5% tween 80 as GUGFAWS-3 the sole source of carbon and energy. Protease and lipase production in the liquid medium has Biont, GUGFAWS-3, grew maximally in the nutrient-rich me- never been reported for genus Halococcus of haloarchaea. dium with 25% crude solar salt as well as in the mineral salt Hence, this finding is of great significance. In nutrient-rich synthetic medium with 20% NaCl with orange-reddish and as well as the synthetic medium, the production of en- mauve pigmentation, respectively. Interestingly, the pigment zymes was growth dependent. Production of protease and had identical spectral features. Such variation in the intensity lipase starts during the early logarithmic phase, reaching a of pigment color is reported in haloarchaeal cultures by Asker maximum at the early stationary phase, and is followed by and Ohta (2002). The total cell mass, however, was reduced a sudden decline as recorded in Fig. 4a, b, respectively. Ann Microbiol (2018) 68:851–861 857

Fig. 2 Characteristics of GUGFAWS-3. a Cell morphology, (i) orange- Fourier transform infrared spectrum (FTIR) of GDEM of GUGFAWS-3 red pigmented colonies, (ii) Gram stain 100X (Nikon H600L), and (iii) and Haloferax alexandrinus KF796625 SEM micrograph. b Extraction efficiency of solvents to pigment. c

This production phase was in agreement with the produc- et al. 2009) and lipase production by Haloarcula sp. G41 tion of protease by Halobacterium sp. SP1(1) (Akolkar (Ozcan et al. 2009).

Fig. 3 Extremozyme potential of bionts GUGFAWS-3, GUGFAWS-2, and GUGFAWS- 3. 1, gelatin; 2, tween 80; 3, starch; 4, carboxy methyl cellulose; 5, xylan; 6, pectin; 7, inulin; 8, DNA; 9, chitin; 10, tannin; 11, ethyl acetate; 12, glutamine; 13, uric acid; 14, agar; and 15, para-nitrophenyl phosphate 858 Ann Microbiol (2018) 68:851–861

Fig. 4 Growth and production of extremozymes by GUGFAWS-3. a Protease. b Lipase. c Protease and lipase. d Stability of lipase. Values are presented as the mean ± standard error (SE) (n =2)

GUGFAWS-3, growing in NTYE with 0.2% tween 80, other haloarchaea (Bhatnagar et al. 2005;Boutaibaetal.2006; exhibited extracellular protease as well as lipase, active at Camacho et al. 2009) and unidentified strains (Ozcan et al. 25% NaCl. Further, as depicted in Fig. 4c, the production of 2009). Constitutive production of lipase is reported by Xin protease and lipase in a single medium, i.e., NTYE with 0.2% and Hui-Ying 2014 in Haloarcula sp. G41. Simultaneous pro- tween 80 followed by the growth of biont and both the en- duction of protease and lipase in the presence of 25% NaCl by zymes, reached a maximum at 96 h. To our knowledge, simul- GUGFAWS-3 is, therefore, the first of its kind. taneous production of protease and lipase has not been report- Our modified assays of Kunitz (1947) and Beisson et al. ed in haloarchaea, although extracellular protease and intra- (2000) for protease and lipase respectively are efficient, repro- cellular lipase which were active at pH 8, 40 °C, in the pres- ducible, functional at 2 M NaCl, and hence recommended for ence of 15–20% NaCl have been reported in extremely halo- monitoring of protease and lipase activity at a high concentra- philic eubacteria Salicola sp. IC10 isolated from hypersaline tion of NaCl. environments (Moreno et al. 2009). Our haloarchaeal strain GUGFAWS-3 is the only haloarchaeon and the only sponge Stability of lipase in the presence of its native biont producing extracellular protease and lipase maximally, protease in 25% NaCl at the stationary phase of its growth. The absence of extracellular lipase in a culture growing in Since both protease and lipase were simultaneously produced NTYE (data not shown) but its presence in the same medium under same optimal conditions, we determined the stability of with tween 80 is indicative of the inducible nature of the lipase lipase in the presence of the protease. Initial lipolytic and pro- enzyme and is unaffected by the presence of protease secreted teolytic activities of cell-free supernatant were 4.42 U mL−1 simultaneously. Inducible lipases are reported only from three and 49.49 U mL−1, respectively. As seen in Fig. 4d, lipase Ann Microbiol (2018) 68:851–861 859 retains 94.07% of its relative activity when stored at 4 °C for compounds such as oligosaccharides, peptides, and fatty acids 24 h. Thus, making the cell-free supernatant containing crude of medicinal applications. Additionally, strain could be used to enzymes highly attractive for detergent formulations. transform marine waste rich in organic moieties to easily metabolizable molecules to be applied in agricultural field. Recently, Gaonkar and Furtado (2018) used immobilized Genotypic analysis of GUGFAWS-3 whole cells of Haloferax ATCC BAA 645 for clarification of proteins and fats in dairy and fish waste water effluents. The 16S rRNA sequence of GUGFAWS-3 showed 99.1% Genus Halococcus has been reported from marine sedi- similarity with Halococcus agarilyticus 62ET and had evolu- ments with low salinities (Braganza and Furtado 2009;Yim tionary similarity of 94.00–99.00% to several available type et al. 2014). Although Haloarchaea sp. has been reported strains of the genus Halococcus (Fig. 5), none of which had from marine sponges by Velho-Pereira and Furtado in 2014, been isolated from sponges. Our GUGFAWS-3 (MF425611) the identification up to genera has not been carried out. isolated from sponge of Haliclona sp. hydrolyzes inulin, Therefore, to the best of our knowledge, this is the first evi- starch, pectin, chitin, tannin, agar, gelatin, tween 80, and ethyl dence of retrieval of biont belonging to genera Halococcus acetate but not urea, DNA, cellulose, xylan, glutamine, and from marine Haliclona, sponge inhabiting the rocky intertidal para-nitrophenyl phosphate (Fig. 3) and simultaneously pro- regions of Anjuna beach, in Goa, India. Intertidal areas are duces protease and lipase extremozymes in a single fermenta- shallow water regions. Those with rocky beds are habitats tion medium containing 25% NaCl. for sponges, gastropods, and other marine invertebrates Of the various reported Halococci, H. sediminicola (Gaitonde et al. 2006; Krediet et al. 2013;Wangetal.2013). CBA1101T (Yim et al. 2014)andH. salifodinae DSM8989T Influx or efflux of water by sponges accumulates large vol- (Wang et al. 2007) hydrolyzed gelatin and tween 80 but not umes of dissolved organic matter (DOC) containing proteins, starch. On the other hand, H. qingdaonensis CM5T and humic acids, lipids of various microbial, and plant and animal H. hamelinensis 100A6T (Wang et al. 2007)hydrolyzedstarch origin. Our success in isolation of Halococcus capable of pro- but not gelatin and tween 80. H. dombrowskii H4T (Wang ducing nine different extracellular enzymes speaks for the role et al. 2007), H. saccharolyticus DSM5350T (Montero et al. of microbial biota in the hydrolysis of DOC through extracel- 1989), and H. morrhuae DSM 1309T (Goh et al. 2006)hydro- lular enzymes, thus providing assimilable carbon to the host lyzed gelatin but not starch and tween 80. Although our strain and also enabling recycling of essential elements in the water GUGFAWS-3 has 99.1% similarity to H. agarilyticus strain bodies (Yahel et al. 2003 and de Goeij et al. 2008). 62ET, the latter unlike to our culture is unable to hydrolyze The present study conclusively reveals (i) the retrieval and tween 80 and starch but has been exploited only for produc- culturing of extremely halophilic Halococcus agarilyticus strain tion of thermo-halophilic agarase (Minegishi et al. 2013, GUGFAWS-3 from Haliclona sp. sponge, inhabiting the inter- 2015). To our knowledge, therefore, H. agarilyticus strain tidal rocky region in Anjuna in Goa, India; (ii) the potential of GUGFAWS-3 is the only Halococcus from sponge strain GUGFAWS-3 for production of nine different hydrolytic Haliclona sp. producing concomitant protease and lipase. enzymes and for simultaneous production of protease and lipase The capability of this strain to utilize diverse polysaccha- in the NTYE medium with 0.2% (v/v) tween 80; and (iii) the rides and proteins as well as fats opens a possibility for biotechnological potential of strain GUGFAWS-3 for production GUGFAWS-3 to be exploited for generation of bioactive of active halophilic protease and lipase extremozymes is of eco- logical and industrial significance.

Acknowledgements S. K. Gaonkar gratefully acknowledges Goa University, research studentship award.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interest.

Fig. 5 Phylogenetic tree of GUGFAWS-3 based on a comparison of 16S References rRNA gene sequences by the neighbor-joining method. The evolutionary distances were computed using the number of differences method and are in the units of a number of base difference per sequence. The analysis Aguiar R, Furtado I (1996) Growth of Halobacterium strain R3on sodium involved 13 nucleotide sequences. Evolutionary analyses were conducted benzoate. In: RS Kahlon (ed) Perspectives in microbiology. Natl. in MEGA 7. Only bootstrap values above 50 were shown Agri Tech Inf Centre India, pp 78–79 860 Ann Microbiol (2018) 68:851–861

Akolkar A, Bharambe N, Trivedi S, Desai A (2009) Statistical optimiza- bacterium Halobiforma sp. strain BNMIITR from Sambhar lake in tion of medium components for extracellular protease production by Rajasthan, India. Ann Microbiol 65:677–686 an extreme haloarchaeon, Halobacterium sp. SP1(1). Lett Appl Hammer Q, Harper DAT, Ryan PD (2001) PAST: paleontological statis- Microbiol 48:77–83 tics software package for education and data analysis. Palaeontol Asker D, Ohta Y (2002) Production of canthaxanthin by Haloferax Electron 4(1):9pp alexandrinius under non-aseptic conditions and a simple, rapid Izotova LS, Strongin AY,Chekulaeva LN, Sterkin VE, Ostoslavskaya VI, method for its extraction. Appl Microbiol Biotechnol 58:743–750 Lyublinskaya LA, Timokhina EA, Stepanov VM (1983) Basu S, Prabhu Matondkar SG, Furtado I (2011) Enumeration of bacteria Purification and properties of serine protease from Halobacterium from a Trichodesmium spp. bloom of the Eastern Arabian Sea: elu- halobium. J Bacteriol 155(2):826–830 cidation of their possible role in biogeochemistry. J Appl Phycol 23: Kamekura M, Seno Y (1993) Partial sequence of the gene for a serine 309–319 protease from a halophilic archaeum Haloferax mediterranei R4 and Beisson F, Tiss A, Riviere C, Verger R (2000) Methods for lipase detec- nucleotide sequences of 16S rRNA encoding genes from several tion and assay: a critical review. Eur J Lipid Sci Technol 102:133– halophilic archaea. Experientia 49:503–513 153 Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee Bhatnagar T, Boutaiba S, Hacene H, Cayol JL, Fardeau ML, Ollivier B, JH, Yi H, Won S, Chun J (2012) Introducing EzTaxon-e: a prokary- Baratti JC (2005) Lipolytic activity from halobacteria: screening and otic 36S rRNA gene sequence database with phylotypes that repre- hydrolase production. FEMS Microbiol Lett 248:133–140 sent uncultured species. Int J Syst Evol Microbiol 62:716–721 Boutaiba S, Bhatnagar T, Hacene H, Mitchell DA, Baratti JC (2006) Krediet CJ, Ritchie KB, Paul VJ, Teplitski M (2013) Coral-associated Preliminary characterization of a lipolytic activity from an extremely microorganisms and their roles in promoting coral health and halophilic archaeon, Natronococcus sp. J Mol Catal B Enzym 43: thwarting diseases. Proc R Soc B 280:20122328 – 21 26 Kunitz M (1947) Crystalline soyabean trypsin inhibitor I general proper- Braganza JM, Furtado I (2009) Isolation of Haloarchaea from low- ties. J Gen Physiol 30:291–310 – salinity coastal sediments and waters of Goa. Curr Sci 96:1182 Manikandan M, Pasic L, Kannan V (2009) Purification and biological 1184 characterization of a halophilic thermostable protease from Camacho RM, Mateos JC, González-Reynoso O, Prado LA, Córdova J Haloferax lucentensis VKMM007. World J Microbiol Biotechnol (2009) Production and characterization of esterase and lipase from 25:2247–2256 – Haloarcula marismortui. J Ind Microbiol Biotechnol 36:903 999 Matsumoto T, Ito M, Fukuda H, Kondo A (2004) Enantioselective Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, Brown CT, transesterification using lipase-displaying yeast whole-cell biocata- Porras-Alfaro A, Kuske CR, Tiedje JM (2014) Ribosomal Database lyst. Appl Microbiol Biotechnol 64:481–485 Project: data and tools for high throughput rRNA analysis. Nucleic Minegishi H, Shimane Y, Echigo A, Ohta Y, Hatada Y, Kamekura M, Acids Res 42:D633–D642 Maruyama T, Usami R (2013) Thermophilic and halophilic β- Dahihande AS, Thakur NL (2017) Differential growth forms of the agarase from a halophilic archaeon Halococcus sp. 197A. sponge Biemna fortis govern the abundance of its associated brittle Extremophiles 17:931–939 star Ophiactis modesta.JSeaRes126:1–11 Minegishi H, Echigo A, Shimane Y, Kamekura M, Itoh T et al (2015) de Goeij JM, van den Berg H, van Oostveen MM, Epping EHG, van Duyl Halococcus agarilyticus sp. nov., an agar-degrading haloarchaeon FC (2008) Major bulk dissolved organic carbon (DOC) removal by isolated from commercial salt. Int J Syst Evol Microbiol 65:1634– encrusting coral reef cavity sponges. Mar Ecol Prog Ser 357:139– 1639 151 Montero CG, Ventosa A, Rodrı’guez-Valera F, Kates M, Moldoveanu N, Dussault HP (1955) An improved technique for staining red halophilic Ruiz-Berraquero F (1989) Halococcus saccharolyticus sp. nov., a bacteria. J Bacteriol 70:484–485 new species of extremely halophilic non-alkaliphilic cocci. Syst Fukushima TT, Usami R, Kamekura M (2007) A traditional Japanese- Appl Microbiol 12:167–171 style salt field is a niche for haloarchaeal strains that can survive in Moreno ML, Garcia MT, Ventosa A, Mellado E (2009) Characterization 0.5% salt solution. Saline Syst 3:2 of Salicola sp. IC10, a lipase- and protease-producing extreme hal- Gaitonde D, Sarkar A, Kaisary S, Silva CD, Dias C, Rao DP, Ray D, ophile. FEMS Microbiol Ecol 68:59–71 Nagarajan R, De Sousa SN, Sarker S, Patil D (2006) Norberg P, Hofsten BV (1969) Proteolytic enzymes from extremely hal- Acetylcholinesterase activities in marine snail (Cronia contracta) – as a biomarker of neurotoxic contaminants along the Goa coast, ophilic bacteria. J Gen Microbiol 55:251 256 West coast of India. Ecotoxicol 15:353–358 Ozcan B, Ozyilmaz G, Cokmus C, Caliskan M (2009) Characterization of Gaonkar SK, Furtado I (2018) Use of immobilized whole cells of extracellular esterase and lipase activities from five halophilic ar- – Haloferax ATCC BAA 645 for treatment of dairy and fish waste chaeal strains. J Ind Microbiol Biotechnol 36:105 110 effluents. IJPBS 8:16–21 Purdy KJ, Cresswell-Maynard TD, Nedwell DB, McGenity TJ, Grant Goh F, Leuko S, Allen MA, Bowman JP,Kamekura M, Neilan BA, Burns WD, Timmis KN, Embley TM (2004) Isolation of haloarchaea that – BP (2006) Halococcus hamelinensis sp. nov., a novel halophilic grow at low salinities. Environ Microbiol 6:591 595 archaeon isolated from stromatolites in Shark Bay, Australia. Int J Raghavan TM, Furtado I (2004) Occurrence of extremely haloarchaea in Syst Evol Microbiol 56:1323–1329 sediments from the continental shelf of west coast of India. Curr Sci – Grant RE (1841) Porifera. Pp 5–9, 310–313, pls II-IV.In: H Bailliere (ed) 86:1065 1067 Outlines of comparative anatomy. 1 London, pp 1–656 Raghavan TM, Furtado I (2005) Expression of carotenoid pigments of – Grant WD (2004) Life at low water activity. Philos Trans R Soc Lond B haloarchaeal cultures exposed to aniline. Environ Toxicol 20:165 359:1249–1267 169 Grant WD, Kamekura M, McGenity TJ, Ventosa A (2001) Order I. Ross HNM, Collins MD, Tindall BJ, Grant WD (1981) A rapid procedure . In: Garrity GM (ed) Bergey’s manual of systematic for the detection of archaebacterial lipids in halophilic bacteria. J bacteriology V.I, The Archaea and deeply branching and Gen Microbiol 123:75–80 phototrophic bacteria, 2nd edn. Springer, New York Shi WL, Zhong CQ, Tang B, Shen P (2006) Purification and characteri- Gupta M, Agarwal S, Navani NK, Choudhury B (2015) Isolation and zation of extracellular halophilic protease from haloarchaea characterization of a protease-producing novel haloalkaliphilic Natrinema sp. R6-5. Acta Microbiol Sin 47(1):163–163 Ann Microbiol (2018) 68:851–861 861

Smibert RM, Krieg NR (1994) In: Gerhardt P (ed) Phenotypic character- Wang X, Brandt D, Thakur NL, Wiens M, Batel R, Schroder HC, Muller ization in: Methods for general and molecular bacteriology. WEG (2013) Molecular cross-talk between sponge host and associ- American Society for Microbiology, Washington DC, pp 607–654 ated microbes. Phytochem Rev 12(3):369–390 Sneath PHA, Mair NS, Sharpe ME, Holt JG (1986) Bergey’smanualof Xin L, Hui-Ying Y (2014) Characterization of an organic solvent-tolerant systematic bacteriology, vol 2. Williams and Wilkins, Baltimore lipase from Haloarcula sp. G41 and its application for biodiesel Velho-Pereira S, Furtado I (2014) Retrieval of euryhaline eubacterial and production. Folia Microbiol 59:455–463 haloarchaeal bionts from nine different benthic sponges: reflection Yahel G, Sharp JH, Marie D, Haese C, Genin A (2003) In situ feeding and of the bacteriological health of waters of Mandapam, India. Indian J element removal in the symbiont-bearing sponge Theonella Mar Sci 43:773–783 swinhoei: bulk DOC is the major source for carbon. Limnol – Ventosa A, Nieto JJ, Oren A (1998) Biology of moderately halophilic Oceanogr 48:141 149 aerobic bacteria. Microbiol Mol Biol Rev 62:504–544 Yim KJ, Cha I, Whon TW, Lee H, Song HS, Kim K, Nam Y,Lee S, Bae J, Vidyasagar M, Prakash S, Litchfield C, Sreeramulu K (2006) Purification Rhee S, Choi J, Seo M, Roh SW, Kim D (2014) Halococcus sediminicola sp. nov., an extremely halophilic archaeon isolated and characterization of a thermostable, haloalkaliphilic extracellular – serine protease from extreme halophilic archaeon Halogeometricum from a marine sediment. Antonie Leeuwenhoek 105:73 79 borinquense strain TSS103. Archaea 2:51–57 Zhao GY, Chen XL, Zhao HL, Xie BB, Zhou BC, Zhang YZ (2008) Hydrolysis of insoluble collagen by deseasin MCP-03 from deep- Wang QF, Li W, Yang H, Liu YL, Cao HH, Dornmayr- Pfaffenhuemer M, sea Pseudoalteromonas sp. SM9913: collagenolytic characters, colla- Stan-Lotter H, Guo GQ (2007) Halococcus qingdaonensis sp. nov., gen binding ability of C-terminal polycystic kidney disease domain, a halophilic archaeon isolated from a crude sea-salt sample. Int J and implication for its novel role in deep-sea sedimentary particulate Syst Evol Microbiol 57:600–604 organic nitrogen degradation. J Biol Chem 283:36100–36107