Defluviitalea phaphyphila sp. nov., a Novel Thermophilic BacteriumThat Degrades Brown Algae

Shi-Qi Ji, Bing Wang, Ming Lu, Fu-Li Li

Shandong Provincial Key Laboratory of Energy Genetics, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy ofSciences, Qingdao, People’s Republic of China

Brown algae are one of the largest groups of oceanic primary producers for CO2 removal and carbon sinks for coastal regions.However, the mechanism for brown alga assimilation remains largely unknown in thermophilic microorganisms. In this work, athermophilic alginolytic community was enriched from coastal sediment, from which an obligate anaerobic and thermophilicbacterial strain, designated Alg1, was isolated. Alg1 shared a 16S rRNA gene identity of 94.6% with Defluviitalea saccharophilaLIND6LT2T. Phenotypic, chemotaxonomic, and phylogenetic studies suggested strain Alg1 represented a novel species of thegenus Defluviitalea, for which the name Defluviitalea phaphyphila sp. nov. is proposed. Alg1 exhibited an intriguing ability toconvert carbohydrates of brown algae, including alginate, laminarin, and mannitol, to ethanol and acetic acid. Three gene clus-ters participating in this process were predicted to be in the genome, and candidate enzymes were successfully expressed, puri-fied, and characterized. Six alginate lyases were demonstrated to synergistically deconstruct alginate into unsaturated monosac-charide, followed by one uronic acid reductase and two 2-keto-3-deoxy-D-gluconate (KDG) kinases to produce pyruvate. Anonclassical mannitol 1-phosphate dehydrogenase, catalyzing D-mannitol 1-phosphate to fructose 1-phosphate in the presenceof NAD�, and one laminarase also were disclosed. This work revealed that a thermophilic brown alga-decomposing system con-taining numerous novel thermophilic alginate lyases and a unique mannitol 1-phosphate dehydrogenase was adopted by thenatural ethanologenic strain Alg1 during the process of evolution in hostile habitats.

Brown algae (Phaeophyceae) are a large group of marine vege-tation that have abundant amounts of the yellow-brown pig-

ment fucoxanthin (1). Their huge biomass makes them one of thelargest oceanic primary producers for CO2 removal and carbonstorage for coastal regions (2). Moreover, as an emerging feed-stock for liquid biofuel production, recently the bioconversion ofbrown algae has been reported frequently (1, 3–6). They have acomplex sugar composition, mainly including alginate, mannitol,and laminarin (6). Alginate is a unique structural polysaccharidein brown algae, and it is abundant in the cell wall for mechanicalprotection (1). It is a linear polysaccharide consisting of twouronic acids, �-L-guluronate (G) and �-D-mannuronate (M) (7).The content of alginate varies by species from 20% to 40% dry cellweight (8, 9). Mannitol is a sugar alcohol form of mannose. Lam-inarin, a storage polysaccharide in many brown algae, is a linearpolysaccharide of �-1,3-linked glucose with small amounts of�-1,6-linkages (10). Mannitol and laminarin are mostly accumu-lated in the summer, and their content could reach as high as 25%and 30% in the species Laminaria hyperborean (11).

The microbial degradation of brown algae involves the decom-position of the structural polysaccharides (mainly alginate andlaminarin) and then the catabolism of the resulting monosaccha-rides (glucose and uronic acid) and mannitol. Glucose was pro-duced by the hydrolysis of glucan and could be assimilated easilythrough glycolysis. Mannitol needs additional steps for catabo-lism before entering glycolysis. In bacteria, the known pathway formannitol assimilation includes two key enzymes (mannitol deg-radation I; MetaCyc Pathway Database [http://metacyc.org/])(12). One is D-mannitol phosphotransferase (PTS) permease,which transports D-mannitol into cells with the formation of D-mannitol 1-phosphate, and the other one is mannitol 1-phosphatedehydrogenase (MPDH), which oxidizes mannitol 1-phosphateto fructose 6-phosphate in a reversible reaction. Fructose 6-phos-

phate then was assimilated through glycolysis. Alginate degrada-tion has been characterized in several bacteria, and the mechanismvaries. For example, Sphingomonas sp. strain A1 can directly trans-port alginate into its cytoplasm through a superchannel, whilemost other bacteria, for example, Zobellia galactanivorans, firstsecrete extracellular alginate lyases to degrade alginate (13, 14).Although strategies differ among bacteria, alginate lyases involvedin the degradation process shared similar catalytic functions. Ac-cording to the classic pathways, alginate first was degraded to oli-goalginate by alginate lyase, and oligosaccharides were furtherexolytically cleaved into unsaturated monosaccharide (spontane-ously rearranged into 4-deoxy-L-erythro-5-hexoseulose uronicacid, or DEH) by oligoalginate lyases (15, 16). Subsequently, DEHwas converted into 2-keto-3-deoxy-D-gluconate (KDG) by DEHreductase. A kinase then catalyzed KDG to 2-keto-3-deoxy-6-phosphogluconate (KDPG), which was directly assimilated throughthe Entner-Doudoroff (ED) pathway. For brown alga degrada-tion, a microorganism should not only possess a whole set of en-zymes, particularly some polysaccharide-degrading enzymes fordepolymerization, but also have a well-evolved redox system to

Received 8 October 2015 Accepted 15 November 2015

Accepted manuscript posted online 20 November 2015

Citation Ji S-Q, Wang B, Lu M, Li F-L. 2016. Defluviitalea phaphyphila sp. nov., anovel thermophilic bacterium that degrades brown algae. Appl Environ Microbiol82:868 – 877. doi:10.1128/AEM.03297-15.

Editor: J. E. Kostka

Address correspondence to Fu-Li Li, lifl@qibebt.ac.cn.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.03297-15.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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balance the reducing equivalents produced from metabolism pro-cesses, especially under anaerobic fermentation conditions (5, 17,18). As far as we know, an integrated system of brown alga degra-dation has never been reported in single natural strains.

Alginate lyases and oligoalginate lyases catalyze the depolymer-ization of alginate into oligomers and monomers through �-elim-ination reactions (19). Their crucial roles in alginate degradation,as well as their biological applications, make them be widelyscreened and studied. Alginate lyases have been isolated from var-ious sources, such as marine algae, marine mollusks, fungi, bacte-ria, and viruses. Most of the characterized alginate lyases werefrom bacteria, including Zobellia, Agrobacterium, Alteromonas,Azotobacter, Bacillus, Enterobacter, Flavobacterium, Klebsiella,Pseudoalteromonas, Pseudomonas, Sphingomonas, and Vibrio (14,19–22). To our knowledge, all of these species are mesophilic bac-teria, and the optimum temperatures of most of the alginate lyasesare below 50°C, with the exception of A1-II (70°C) from Sphin-gomonas sp. strain A1 (23) (http://www.brenda-enzymes.org/index.php).

In previous work, we demonstrated that the coastal marineenvironment harbored a diversity of thermophilic cellulolyticbacteria (24). These bacteria showed extremely low 16S rRNAgene identities to their closest relatives, indicating an untappedthermophilic microbial resource. In this work, by using kelp pow-der as a carbon resource, we enriched the same sediment samplecollected from Qingdao coast, and a novel bacterial strain, Alg1,was isolated. Community survey and strain characterization wereconducted, and an integrated brown alga-degrading system inAlg1 was revealed based on genome analysis and key enzyme ver-ification.

MATERIALS AND METHODSEnrichment protocol and survey of enriched community. Samples werecollected from marine sediment of a coastal region of the Yellow Sea(36°5=N, 120°32=E), China, in May 2013. One gram of sediment was usedas the inoculum in 100 ml of basal medium (BM) at an initial pH of 7.4and containing 1 g of kelp (Saccharina japonica) powder as the carbonsource at 60°C under anaerobic conditions. BM consisted of 0.1 g/liter ofKH2PO4, 0.1 g/liter of K2HPO4, 1 g/liter of NaHCO3, 2 g/liter of NH4Cl,30 g/liter sea salt, 0.5 g/liter of L-cysteine, 1 g/liter yeast extract, and 0.0001(wt/vol) resazurin. Vitamins were added at the following concentrations(in milligrams per liter): pyridoxamine dihydrochloride, 1; p-aminoben-zoic acid (PABA), 0.5; D-biotin, 0.2; vitamin B12, 0.1; thiamine-HCl-2H2O, 0.1; folic acid, 0.2; pantothenic acid calcium salt, 0.5; nicotinic acid,0.5; pyridoxine-HCl, 0.1; thioctic acid, 0.5; riboflavin, 0.1.

Cultures growing in the presence of kelp were transferred 10 times toget a relatively stable community. The community was surveyed by con-structing a 16S rRNA gene clone library as described previously (24). PCRamplifications targeting the 16S rRNA gene used the universal oligonu-cleotide primers 27F and 1492R (see Table S1 in the supplemental mate-rial), and the PCR amplicons were cloned into a pMD18T vector. Twovector-specific primers (M13-47 and RV-M) (see Table S1) were used forthe amplification and verification of the DNA inserts.

Strain isolation and characterization. Strain isolation from the com-munity was carried out by plating the serially diluted consortium cultureon an anaerobic agar plate containing 0.5% alginate in BM (ABM) with1.5% agar in an anaerobic chamber. The temperature, salinity, and pHranges for cell growth were determined in ABM by following the methodsof Wang et al. (25). The optical density at 600 nm (OD600) was used to testcell growth for determining the optimal temperature, salinity, and pH forstrain Alg1. The utilization of the following substrates as carbon and en-ergy sources was tested in BM with a concentration of 0.5% (wt/vol):acetate, starch, pyruvate, ribose, fructose, lactate, glucose, maltose, xylose,

peptone, lactose, galactose, mannose, raffinose, sucrose, arabinose, cello-biose, glycerol, mannitol, rhamnose, peptone, Casamino Acids, lami-narin, and alginate.

The cell shape of Alg1 was observed using a scanning electron micro-scope (S-4800; Hitachi, Tokyo, Japan) (25). Chemotaxonomic character-istics were determined from cells grown at 60°C for 2 days in BM contain-ing 0.5% mannitol. Fatty acids were extracted and analyzed according tothe standard protocol of the MIDI (Microbial Identification) system. TheG�C content of the DNA was determined by using high-performanceliquid chromatography (HPLC) (Waters, Milford, MA) (26). The con-centrations of fermentation products were analyzed by HPLC using anAminex HPX-87H column (Bio-Rad, Hercules, CA).

Phylogenetic analyses. For the 16S rRNA gene clone library, DNAStarLasergene software was used for manual editing of the amplified 16SrRNA gene sequences. The definition of operational taxonomic units(OTU) at 97% sequence identity was determined using the DOTUR soft-ware package (27). The identification of phylogenetic neighbors and thecalculation of pairwise 16S rRNA gene sequence identities were achievedby a BLAST search in the EzTaxon-e database and nucleotide databases ofthe National Center for Biotechnology Information (NCBI) (28). Phylo-genetic analysis was performed by the software package MEGA, version5.0, after multiple alignment of data by CLUSTALX (29). The phyloge-netic tree was constructed using neighbor-joining (NJ) methods.

Genome sequencing and sequence analysis. Genomic DNA of strainAlg1 was prepared by following a procedure described previously (30).The genome was sequenced using the Illumina HiSeq 2000 system afterconstructing the Illumina paired-end DNA library with 170- and 500-bpinserts. The numerous reads were assembled into hundreds of contigs byVelvet (V1.2.03), which were resorted to predict gene functions conse-quently using Glimmer, GeneMark, and Zcurve. The genes were anno-tated through NCBI, KEGG, and SEED databases, classified through theCDD database, and constructed into metabolic pathways through theKEGG database. Signal peptides were predicted with SignalP 4.1 (31).Conserved domains within a coding nucleotide sequence were analyzedusing CD-search (32).

DNA sequence analysis of the three gene clusters involved in brownalga degradation was performed to identify patterns that mediate tran-scription, i.e., promoters and Rho-independent terminators, by using theBPROM online program and ARNold online program (33–35).

Enzyme expression, purification, and identification. For the purifi-cation of the enzyme encoded by dp0100, strain Alg1 was grown to sta-tionary phase in BM supplemented with 1% alginate. Cells and residualalginate were removed by centrifugation for 20 min at 10,000 � g and 4°C.The supernatant was passed through a 0.22-�m filter, brought to the samevolume of cold (�20°C) acetone, and centrifuged for 20 min at 12,000 � g.The precipitate was resuspended with 7 ml 50 mM Tris-HCl (pH 7.3)(buffer A) and was dialyzed using a dialysis bag (Solarbio, Beijing, China)with a permeability molecular weight range of 8,000 to 14,000 in buffer A.The dialyzed protein was injected into an anion exchange column (HiPrep16/10 Q FF; 20 ml; GE Healthcare, Little Chalfont, United Kingdom)equilibrated with the same buffer. Proteins were eluted at 5 ml min�1 witha 400-ml linear gradient of 0 to 1 M NaCl in buffer A. Each fraction (15 ml)was assessed for alginate lyase activity. An active fraction was obtained ataround 300 mM NaCl and then was loaded onto a gel filtration column(HiPrep 16/60 and Sephacryl S-200 HR; GE Healthcare) and eluted withbuffer A. The eluted fractions were analyzed by SDS-PAGE. All chroma-tography procedures were performed on an ÄKTA purifier system (GEHealthcare). The purified proteins were identified by liquid chromatog-raphy-tandem mass spectrometry (LC-MS/MS) analysis (36). All putativebrown alga degradation-related genes except dp0100 were selected forheterogeneous expression by following the method of Zhang et al. (36).

Enzyme activity assays. Alginate lyase activity was assayed by measur-ing the increase in absorbance at 235 nm (A235) of the reaction products(unsaturated uronates) for 3 min at 65°C in a quartz cuvette containing 2ml of 0.2% alginate in 100 mM acetate-sodium acetate buffer (pH 5.8) and

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10 �l purified enzymes. One unit of activity was defined as an increase of0.1 in the A235 per minute. Laminarinase activity was assayed by incubat-ing laminarin solution (1%, wt/vol, in buffer A, pH 7.3) with purifiedenzyme at 65°C for 3 min. The release of reducing sugars was measured bythe dinitrosalicylic colorimetric (DNS) method (37). One unit of lami-narinase activity was defined as the amount of enzyme that released 1�mol of reducing sugar per min. Protein concentrations were determinedby using the Bradford assay kit (Biomed, Beijing, China) with bovineserum albumin as the standard (38).

The measurement of 2-keto-3-deoxy-D-gluconate 6-dehydrogenase(KdgD) activity followed the method previously described (14). The sub-strate DEH was produced by the exotype alginate lyase Dp1761E. Twohundreds microliters of purified Dp1761E (367 �g ml�1) was added to an800-�l reaction mixture containing 0.5% (wt/vol) alginate sodium saltand 100 mM acetate-sodium acetate buffer (pH 5.8). After incubation at60°C for 24 h, the mixture was centrifuged at 12,000 � g for 30 min andthe supernatant was conserved as the DEH solution. The reaction mixturefor the KdgD assay consisted of 50 �l of purified Dp1759E (28 �g ml�1),200 �l of DEH solution, 100 �l of NADH (1 mM), and 400 �l potassiumphosphate buffer (50 mM, pH 6.0). The enzyme kinetic was determined at60°C, and activity was monitored by measuring the decrease of the absor-bance at 340 nm (A340). KDPG aldolase (KdpgA) activity was determinedby using an enzyme-coupled spectrophotometric assay (14). KDG wasproduced from DEH in the same reaction system as that mentionedabove. After 30 min at 60°C, the reaction was stopped by heating themixture at 100°C for 5 min. After cooling to 60°C, 50 �l of Dp1703E (176�g ml�1) or Dp1704E (189 �g ml�1) and 20 �l ATP (100 mM) wereadded to the reaction for another 30 min to allow the formation of ADP.The amount of ADP in the solution was measured by a coupling enzymeassay at 35°C containing 50 mM potassium phosphate buffer (pH 7.4), 5mM MgCl2, 1 mM phosphoenol pyruvate, 0.3 mM NADH, and 1.5 Ulactate dehydrogenase–1.0 U pyruvate kinase (both from rabbit muscle).The reaction was initiated by adding 200 �l ADP-containing reactionmixture. The reduction of fructose 1-phosphate (F1P; �98% purity; Car-bosynth Ltd., Berkshire, United Kingdom) and fructose 6-phosphate(F6P; �98% purity; Sigma-Aldrich, St. Louis, MO) by MPDH was assayedin 800 �l sodium phosphate buffer (50 mM, pH 6) with 0.2 mM NADHand 50 �l of Dp0124E (149 �g ml�1). F1P or F6P (1 mM) was used toinitiate the reaction. MPDH activities were determined from the rate ofNADH oxidation at 60°C by measuring the absorbance at 340 nm. Thereversible reaction would indicate the oxidation product from mannitol1-phosphate. For Dp1703E, Dp1704E, Dp1759E, and Dp0124E, 1 U ofactivity was defined as 1 �mol NADH oxidized or produced per min.

Accession numbers. The nucleotide sequences of 16S rRNA genesfrom the clone libraries have been deposited in the GenBank databaseunder accession numbers KF775581 to KF775589 and KJ411294 toKJ411335. The accession number of the 16S rRNA gene sequence ofAlg1 is KJ411293. The whole-genome shotgun project of Alg1 hasbeen deposited at DDBJ/EMBL/GenBank under the accession numberJWID00000000. The version described in this paper is versionJWID01000000.

RESULTSSurvey and isolation of the community. From the generatedclone library of the enriched kelp-degrading community, a total of52 clones from the 80 positive transformants were verified as 16SrRNA genes and were used for OTU analysis. Eleven OTUs werefound to be represented in the clone library under the definitionof 97% sequence identity (Fig. 1). OTU1 to OTU8 represented94% (49/52) of the sequenced clones and were classified in onelarge branch with one uncultured species from Arctic sediment(GenBank accession number FN396782), which together formeda parallel branch with Defluviitalea saccharophila LIND6LT2T andsome uncultured species from various thermophilic sources (39,

40). OTU9 and OTU10 were closely related to an extremely alka-liphilic bacterium, Alkaliphilus transvaalensis, isolated from adeep South African gold mine (41). OTU11 was found to be closeto an uncultured bacterium of clone 27 (GenBank accession num-ber JQ741987) from our former research of a thermophilic cellu-lolytic community, with 16S rRNA gene identities of 95% (24). Allof the clones from the brown alga-degrading community be-longed to the order Clostridiales.

Dozens of colonies with uniform size were formed on the agarplate in the process of strain isolation. Individual colonies werepicked up for examination, and all of them shared very similar 16SrRNA gene sequences with the clones in OTU1 (data not shown).One isolate, designated strain Alg1, was used for further study.

Strain characterization. Strain Alg1 was an obligate anaerobicand Gram-negative bacterium with a fermentative metabolism,forming a translucent colony on plates. Alg1 can utilize the maincomponents of brown algae, including alginate, mannitol, andlaminarin, as sole carbon sources with main products of ethanoland acetic acid (Table 1) and can utilize fructose and ribose butnot sucrose and xylose (Table 2). The growth temperature wasfrom 45°C to 65°C, with the optimum around 55 to 60°C. Alg1 didnot show any growth with NaCl instead of sea salt. The toleranceof salinity was from 1 to 5%, with the optimal salinity at 3%. ThepH tolerance was from pH 6 to 9, and the optimum pH was be-tween 7 and 8. The cell morphology of Alg1 was rod-shaped, withdifferences in alginate medium (long rod) and in mannitol me-dium (short rod) (Fig. 2). The cellular fatty acid composition ofthe strain Alg1 was analyzed. The major fatty acids were C16:0

(63%), C17:1 iso w5c (24%), and C18:0 (5.2%). The DNA G�C con-tent of strain Alg1 was 28 mol%.

The 16S rRNA gene identification demonstrated that the clos-est neighbors of strain Alg1 with validly published names werefrom the order Clostridiales in the phylum Firmicutes, with 94.6%and 89.3% sequence identities to D. saccharophila LIND6LT2T

and Vallitalea guaymasensis Ra1766G1(T), respectively (42). AnNJ phylogenetic tree based on the 16S rRNA gene revealed thatstrain Alg1 clustered with D. saccharophila but with a clear phylo-genetic distinction, indicating that Alg1 is a new member of thegenus Defluviitalea (Fig. 3). Based on the phenotypic, chemotaxo-nomic, and phylogenetic distinctiveness, strain Alg1 is consideredto represent a novel species of the genus Defluviitalea, named De-fluviitalea phaphyphila. The type strain is Alg1T (the same as CG-MCC 1.5199T and JCM 30481T).

Genome analysis of genes responsible for brown alga degra-dation. After assembly and mapping of the sequenced data fromthe constructed clone libraries of the genomic DNA of strain Alg1,a total of 76 scaffolds containing 129 contigs with a length of 2.54Mb were obtained. The G�C content of the genome is 28%,which was consistent with the results of strain characterization.

Through genome analysis, three gene clusters and numerouskey genes involved in alginate, laminarin, and mannitol degrada-tion were found (Fig. 4). One of the most important phenotypicproperties of strain Alg1 is the ability to depolymerize alginateunder thermophilic conditions. Genome analysis revealed sixgenes encoding alginate lyases (Table 3). Dp0084 was one of thepredicted alginate lyases with an N-terminal secretion signal,which contained a polysaccharide lyase (PL) 6 family domain(cd14251) conserved in both alginate lyase (EC 4.2.2.3) and chon-droitinase B (EC 4.2.2.19). Dp0100, Dp1059, Dp1761, andDp1770 were predicted to contain a heparinase II/III-like protein

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FIG 1 Phylogenetic analysis of the clones from the constructed 16S rRNA gene library and their relatives from the GenBank database after multiple alignmentsof data. The numbers in brackets represent the number of clones in the OTU.

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domain (pfam07940) (Table 3), which was found in both hepari-nase II and alginate lyase (43, 44). Dp2072 was predicted to con-tain an alginate_lyase 2 domain (pfam08787) and shared the high-est amino acid identity (52%) with the alginate lyase fromCorynebacterium species (45). Dp0100 and Dp2072 both carry anN-terminal secretion signal, as predicted by SignalP 4.1, and arebelieved to be extracellular alginate lyases. Dp0100 is a multido-main protein, and its catalytic domain (heparinase II/III-like pro-tein domain) shared 18% identity with exotype alginate lyaseAtu3025 from Agrobacterium tumefaciens (22). Other domains allwere predicted as the substrate binding domains. Dp1761 shared21% identity with an exo-oligoalginate lyase of PL 17 from Sac-charophagus degradans 2-40 (46), and Dp1770 was closely relatedto an alginate lyase of PL 15 from Agrobacterium tumefaciens C58(22). Both Dp1761 and Dp1770 contained no signal sequences;therefore, they were hypothesized to be intracellular oligoalginatelyases. In addition to alginate lyases, Dp1703, Dp1704, Dp1705,and Dp1759 also were believed to play important roles in alginateutilization according to genome analysis. Dp1759 was predicted tobe a DEH reductase and shared 38% amino acid identity with anNADH-dependent reductase of Sphingomonas sp. strain A1 (47).A small gene cluster including dp1703-dp1705 was involved in theconversion of KDG to pyruvate (Fig. 4B). Dp1703 and Dp1704both were predicted to have a conserved domain of 2-keto-3-deoxyglucononate kinase (KdgK), while Dp1705 contained a con-served domain of KdpgA (Table 3).

Genes for mannitol and laminarin metabolism also were ana-lyzed. The predicted genes involved in mannitol catabolism werefound to be located in gene cluster I, which included dp0124-dp0126 (Fig. 4B). Dp0126 was annotated as a mannitol-specifictransporter and shared 64% identity with the PTS system manni-tol-specific transporter subunit IICBA from Alkaliphilus metalli-redigens. Dp0124 contained a medium-chain reductase/dehydro-

genase (MDR) domain and was hypothesized to be MPDH.Dp0125 contained a FruK_PfkB_like domain (cd01164) andshared a high identity of 53% with a 1-phosphofructokinase(PFK1) from Alkaliphilus metalliredigens; thus, it was consideredPFK1. Laminarin can be hydrolyzed readily by glucosidases. Twoglucosidase-encoding genes were annotated through genomeanalysis, dp0614 and dp1433. Dp0614 displays an N-terminal se-cretion signal and contains a GH16_laminarinase_like domain,which shares 47% amino acid sequence identity with the glucanendo-1,3-beta-D-glucosidase (EC 3.2.1.39). Dp1433 shared 52%amino acid sequence identity with �-glucosidase (EC 3.2.1.21)and had no secreting signal.

Biochemical characterization of the key enzymes involved inbrown alga degradation. Genome analysis revealed at least 15genes involved in brown alga degradation, which included 10alginate degradation-related genes, 3 mannitol utilization-re-lated genes, and 2 laminarin degradation-related genes (Table3). dp0100 has a nucleotide sequence of 5,478 bp and is difficult toexpress heterogenously. Thus, direct protein purification from thefermentation supernatant of Alg1 was conducted. Alginate lyasesin the culture supernatant were purified with an anion exchangecolumn followed by a filtration column, and two absorption peakswere detected for alginate lyase activity. Two peak fractions (A andB) were digested with trypsin and were identified by mass spec-trometry. These peptides perfectly matched Dp2072 and Dp0100,with estimated molecular masses of 37 kDa and 205 kDa and cov-ering 69% and 68%, respectively, of their whole sequences (seeFig. S2 in the supplemental material). Heterogenous expressionwas applied for all other genes except dp0100. Ten recombinantenzymes were successfully expressed in a soluble form, whereasDp1433 and Dp1705 failed to form soluble proteins in Escherichiacoli BL21(DE3) (see Fig. S1). Dp0084, Dp0100, Dp1059, Dp1761,Dp1770, and Dp2072 were found to be able to degrade alginate,with specific activities of 3,592, 2,850, 9, 58, 5, and 210 U mg�1

(Table 3), respectively. Moreover, Dp1761 was confirmed to act inexo mode on alginate, and DEH released by Dp1761 was furtherused as the substrate to evaluate the activity of the putative DEHreductase Dp1759E. After the addition of purified Dp1759 to thereaction mixture, an immediate decrease of absorbance at 340 nmwas observed, indicating the oxidation of NADH (see Fig. S3).Therefore, Dp1759 was confirmed as a KdgD member catalyzingDEH to KDG. The kinase activities of Dp1703 and Dp1704 weredetected using KDG produced by Dp1759 as the substrate. Afterthe enzyme-coupled assay system was initiated, significant en-zyme reactions were observed as decreases in the A340 for both

TABLE 2 Physiological, biochemical, and chemotaxonomic characteristics of Alg1

Parameter

Result for:

Alg1 D. saccharophila

Gram stain Negative NegativeOptimal temp (°C) 55–60 50–55Optimal salinity (%) 3 0.5Optimal pH 7.5 7–7.5G�C content (mol%) 28 35.2Carbon source utilization Ribose, fructose, glucose, mannitol, cellobiose,

laminarin, alginateGlucose, xylose, sucrose, cellobiose, mannitol

Fermentation products Ethanol acid, acetic acid Acetic acid, formic acid, butyric acidMajor fatty acids C16:0 (63%), C17:1 iso w5c (24%), C18:0 (5.2%) C16:0 (68.4%), C14:0 (8.3%), C18:0 (7.3%)

TABLE 1 Ethanol and acetic acid production of Alg1 from alginate,mannitol, and glucose after 120 h of fermentation

Carbon source(2%, wt/vol)

Production (g/liter) ofa:

EthanolAceticacid

Remainingsugars

Alginate 3.5 0.3 3.2 0.2 4.5 1.4Mannitol 7.0 0.5 0.18 0.1 0.4 0.3Glucose 6.4 0.1 0.8 0.1 0a Results are the means (with standard deviations) from three independentexperiments.

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Dp1703 and Dp1704 (see Fig. S3). Consequently, the conversionof KDG to KDPG was phosphorylated by Dp1703 and Dp1704.Dp0614 showed high hydrolyase activity (95 U mg�1) againstlaminarin and was confirmed as an active laminarinase.

For the measurement of the dehydrogenase activity ofDp0124, no reaction was observed in the presence of F6P. Incontrast, after the addition of F1P, the absorbance at 340 nmobserved was significantly decreased with the oxidation ofNADH and the formation of NAD� (see Fig. S3 in the supple-

mental material). This result indicated that F1P is the oxidationproduct from mannitol 1-phosphate.

DISCUSSION

The alginolytic community was enriched from a coastal sedi-ment sample at low temperature, and it showed a relativelyhomogeneous bacterial structure compared to the structure ofa cellulolytic community we previously identified (24). Wefound the members of the alginolytic community all were from

FIG 2 Cell morphologies of Alg1 by scanning electron microscopy (SEM), utilizing alginate (A) and mannitol (B) as carbon sources.

FIG 3 Phylogenetic tree of the 16S rRNA gene of Alg1 and its closest related species from GenBank. Bootstrap values were calculated based on 1,000 repli-cates.

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the order Clostridiales, and a high proportion of the clones (94%;49/52) was phylogenetically related to D. saccharophilaLIND6LT2T. In contrast, only 76% of the clones of the cellulolyticcommunity were from Clostridiales, and 62% of the clones werephylogenetically related to the predominant species (24), suggest-ing the deconstruction of lignocellulose is a more complex processthan that of brown algae. To our surprise, all of the species fromboth communities turned out to be novel species with low 16S

rRNA gene sequence (95%) identities to their closest relatives.The thermophilic properties of these species raised the interestingtopic of the distribution of marine thermophiles, which normallywere found to be widely distributed in high-temperature habitats,including deep-sea hydrothermal vents, subsurface petroleumreservoirs, and hot springs. Increasing amounts of experimentalevidence have indicated that the distribution of thermophiles isnot limited to such geothermal areas. For example, Hubert et al.

FIG 4 Putative pathways and gene clusters involved in brown alga degradation deduced from the genome of Alg1. (A) Predicted pathways participating in brownalga degradation. (B) Three gene clusters involved in deconstruction of brown algae in the genome of Alg1. The functions of the proteins are color-coded: green,alginate degradation-related genes; yellow, mannitol and fructose metabolism-related genes; black, putative regulation factor; blue, unknown function. PI1, PII1,PII2, PII3, PIII1, PIII2, PIII3, and PIII4 are predicted promoters, and TI, TII, and TIII are predicted Rho-independent terminators. SDR, short-chaindehydrogenase/reductase.

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demonstrated the diversity of dormant thermophilic bacterialspores that become active at much higher temperatures than theydo in situ in Arctic marine sediments (40, 48). The thermophilicalginolytic and cellulolytic communities, which were enrichedfrom coastal low-temperature environments, have provided ex-citing evidence for the wide distribution of thermophiles (24),suggesting untapped novel microbial resources exist in coastal en-vironments.

Hubert et al. explained the origins of the thermophiles in Arcticsediment with a theory that thermophiles from warm subsurfacepetroleum reservoirs and ocean crust ecosystems distribute intothe cold ocean through seabed fluid flow (40). From the phyloge-netic analysis of the alginolytic community, some clones showedhigh 16S rRNA gene sequence identities of 95% to 99% to theclones from Arctic sediment, suggesting these clones shared sim-ilar origins from marine thermal ecosystems. Moreover, thecoastal thermophilic species also showed close proximity to spe-cies from some terrigenous environments, including hot springs,deep mines, and oil fields, which suggested the involvement ofother origins (24). Until now, few thermophilic bacterial purecultures have been isolated from low-temperature marine envi-ronments. Alg1, as one of the few representatives, may give ussome direct information on this mysterious population.

Alg1 is a member of spore-forming species from the phylumFirmicutes. The vital requirement of sea salt and the versatile abil-ity in degrading brown algae observed in Alg1 indicate that it is areal marine bacterium that has adapted to the nutritional condi-tions of the marine environment. Taken together, these resultsconveyed an inspiring message that novel marine thermophileswith functional and metabolic diversities could be acquired fromthe coastal environment. The genome analysis and biochemicalcharacterization of key enzymes involved in the deconstruction ofbrown algae demonstrated that Alg1 contains a full set of genesparticipating in the catabolism of brown algae (Fig. 4). Moreover,the main products from brown alga catabolism were ethanol andacetic acid. To our knowledge, Alg1 is the first reported fermen-tative natural bacterium capable of degrading brown algae.

As an anaerobic and thermophilic bacterial strain, Alg1 was

determined to have the ability to utilize alginate, a characteris-tic mostly found in mesophilic bacteria. Two gene clusters andnumerous other genes were found to be directly involved in themetabolism of alginate (Fig. 4). dp1759, dp1761, and dp1770were located in gene cluster III, which was predicted to playimportant roles in the intracellular metabolism of alginate(from oligoalginate to KDG). Three carbohydrate transporter-encoding genes (dp1764, dp1768, and dp1769) were assumed totransport oligoalginate to the cytoplasm, while their functionsneed further experimental verification. Furthermore, clusterIII was predicted to be a transcriptional unit containing fourpromoters upstream and one terminator downstream (see Ta-ble S2 in the supplemental material). A complex operon likethis could ensure the genes located in the cluster closely coop-erate for oligoalginate assimilation. Cluster II also was pre-dicted as an operon with multiple promoters. Dp1703 andDp1704, two KdgK members, showed only 29% identity toeach other, indicating different origins. A similar situation wasfound in the marine alginolytic bacterium Z. galactanivorans,and two KdgK genes (zg4703 and zg2614) were characterizedfrom its genome (14). Dp1705 was predicted to encode KdpgA,which catalyzes KDPG to pyruvate and glyceraldehyde 3-phos-phate (G3P) in the ED pathway. The enzymes in cluster II playcrucial roles in the complete degradation of alginate into pyru-vate, and the predicted multiple promoters could regulate theexpression level of individual genes more flexibly to meet thenecessities of the cells. The enzymatic degradation of laminarinwas common in quite a few of microorganisms, including bac-teria, actinomycetes, and fungi (10), and its degradation path-way is much simpler than that of alginate (Fig. 4A). Dp0614 canendolytically cleave laminarin into oligolaminarin, which wasfurther degraded to glucose by a predicted �-glucosidase(Dp1433).

After the cell structure of brown algae was disrupted by thesecreted alginate lyases and laminarinase, mannitol was re-leased into the medium. Although mannitol could be utilizedby a number of microorganisms, it cannot be fermented under

TABLE 3 Key genes of the proposed pathways for alginate, laminarin, and mannitol degradation and enzyme activities

Genename Annotated function based on conserved domain

Encodedproteinsize (kDa)

Enzymecharacteristica Substrate

Assaytemp (°C)

Sp act(U/mg)

dp0084 Lyase 52 Exp Alginate 65 3592dp0100 Heparinase II/III-like protein 205 Pur Alginate 65 2850dp1059 Heparinase II/III-like protein 75 Exp Alginate 65 9dp1761 Heparinase II/III-like protein 73 Exp Alginate 65 58dp1770 Heparinase II/III-like protein 77 Exp Alginate 65 5dp2072 Alginate lyase 37 Exp Alginate 65 210dp1703 2-Keto-3-deoxygluconokinase 34 Exp KDG 60 0.4dp1704 2-Keto-3-deoxygluconate kinase 38 Exp KDG 60 2.4dp1705 2-Keto-3-deoxyphosphogluconate aldolase 23 Pre KDPG —b —dp1759 Short-chain dehydrogenase/reductase (SDR) 27 Exp DEH 60 6.9dp0124 Medium-chain reductase/dehydrogenase (MDR) 46 Exp Mannitol 1-phosphate 60 4.3dp0125 1-Phosphofructokinase 34 Pre Fructose 1,6-bisphosphate — —dp0126 PTS system mannitol-specific transporter subunit IIC 67 Pre Mannitol — —dp0614 GH16_laminarinase-like domain 73 Exp Laminarina 65 95dp1433 Beta-glucosidase 54 Pre Glucan — —a Exp, enzyme is heterologously expressed; Pur, enzyme is purified from the native strain; Pre, enzyme is predicted through bioinformatics analysis.b —, not detected.

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strictly anaerobic conditions by some ethanol-producing mi-croorganisms, such as Zymobacter palmae and Saccharomycescerevisiae (49). This is attributed mainly to the excess electrons(NADH) produced during mannitol metabolism. Under anaero-bic conditions, these excess reducing equivalents could not beneutralized by oxygen, especially when the microorganisms lacktranshydrogenase (an enzyme converting the catabolic reducingequivalent NADH to the anabolic reducing equivalent NADPH)(17, 49). The vigorous growth of Alg1 using mannitol as the car-bon substrate under obligate anaerobic conditions indicated thatAlg1 has a well-balanced system to process the excess reducingequivalents. However, the precise regulation for reducing equiva-lents still needs further investigation. A mannitol-specific PTS sys-tem encoded by dp0126 is responsible for the transport of manni-tol into cytosol, and mannitol is converted to mannitol1-phosphate during this process (Fig. 4A). MPDH encoded bydp0124 then converts mannitol 1-phosphate to fructose 1-phos-phate with the production of one NADH molecule. Fructose1-phosphate was phosphorylated by PFK1 (Dp0125) to producefructose 1,6-bisphosphate, which was further assimilated throughglycolysis (Fig. 4A). In contrast to the classical mannitol degrada-tion pathway found in bacteria in which mannitol 1-phosphatewas converted into fructose 6-phosphate, the mannitol degrada-tion pathway possessed by Alg1 showed distinct features in con-verting mannitol 1-phosphate to fructose 1-phosphate. Actually,Dp0124 first was annotated as an L-sorbose 1-phosphate reduc-tase; however, no activity was detected in the presence of D-sorbi-tol 6-phosphate and NAD(P)� (data not shown). After the forma-tion of pyruvate from the glycolysis and ED pathways inmetabolizing alginate, laminarin, and mannitol, ethanol synthesiswas initiated.

Taxonomy. Defluviitalea phaphyphila (pha.phy. phi’la. Gr. n.phaiós-ophyta, brown algae; N.L. adj. philus -a -um [from Gr. adj.philos -ê -on], friend, loving; N.L. fem. adj., phaphyphila, brownalgae-loving).

Cells are Gram-negative, rod-shaped, 0.4 to 0.5 �m by 1 to5 �m. Forming half-transparent colony on ABM agar plate.Obligately anaerobic with fermentative metabolism. Utilize al-ginate, mannitol, laminarin, fructose, glucose, mannose, cello-biose, and ribose but not sucrose, xylose, acetate, starch, pyru-vate, ribose, fructose, lactate, maltose, xylose, peptone, lactose,galactose, raffinose, arabinose, glycerol, rhamnose, peptone,and Casamino Acids. Growth temperature covers from 45 to65°C, with an optimum at 55 to 60°C. Alg1 did not show anygrowth with NaCl instead of sea salt. The tolerance of salinitywas from 1 to 5%, with an optimal salinity of 3%. The pHtolerance was from pH 6 to 9, and the optimum pH was be-tween pH 7 and 8. The major fatty acids were C16:0 (63%),C17:1 iso w5c (24%), and C18:0 (5.2%). The G�C content of thegenomic DNA in strain Alg1 was 28 mol%. The type strain isAlg1 (the same as CGMCC 1.5199T and JCM 30481T), whichwas isolated from coastal sediment of an amphioxus breedingzone in Qingdao, China (36°5= N, 120°32= E).

ACKNOWLEDGMENTS

We thank Chen Li from the Yellow Sea Fisheries Research Institute, Chi-nese Academy of Fishery Sciences, for her help with determination of thecellular fatty acids.

FUNDING INFORMATIONNational Natural Science Foundation of China (NSFC) provided fundingto Shi-Qi Ji under grant number 41506155. Science and Technology De-velopment Project of Shandong Province provided funding to Ming Luunder grant number 2014GHY115027. Shandong Province Natural Sci-ence Funds for Distinguished Young Scholar provided funding to Fu-Li Liunder grant number JQ201507. Qingdao Institute of Bioenergy and Bio-process Technology Director Innovation Foundation for Young Scientistsprovided funding to Shi-Qi Ji under grant number Y37207210B.

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