The Family Halomonadaceae

Abstract

The family Halomonadaceae, within the class Gammaproteobacteria, consists mostly of marine and moderately halophilic microorganisms that are phenotypically rather diverse. As of January 2014, this family contains ten genera and 106 validly published species names and, therefore, constitutes the largest group of halophilic bacteria. In this chapter, the historical and current taxonomy have been reviewed along with molecular and phenotypic analyses. In addition, isolation and preservation procedures were considered, as well as the ecological habitats where the members of the family Halomonadaceae can be found and their clinical relevance as human pathogens. The increasing interest of this group of microorganisms due to its biotechnological and environmental applications has also been addressed.

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Introduction

The family Halomonadaceae, within the class Gammaproteobacteria, consists mostly of marine and moderately halophilic microorganisms that are phenotypically rather diverse. Because of this apparent lack of a core of differential phenotypic traits, many of its current species were previously assigned to other genera such as Deleya (now extinct), Alcaligenes, Pseudomonas, Halovibrio, Volcaniella, etc. Reorganizations among these species started by the mid 1990s with the aid of 16S rRNA gene sequence comparison. In the meanwhile, new genera and species descriptions within the family Halomonadaceae have been reported, and the increasing number of species led some authors to review its phylogeny (Arahal et al. 2002c; de la Haba et al. 2010a, 2012) and phenotypic features (Mata et al. 2002).

A Subcommittee on the Taxonomy of the Halomonadaceae, a member of the International Committee on Systematic of Prokaryotes, was constituted more than 10 years ago (Vreeland and Ventosa 2003) and can be taken as a sign of the increasing interest in this group of organisms.

Species of the genera Halomonas and Chromohalobacter have been largely studied as model organisms of halophilism. Some of their representatives are among the most halophilic bacteria (Ventosa et al. 1998) and are adapted to a wide range of saline concentrations, even wider than extreme halophiles. Another source of interest for the study of this group of organisms has been their potential in biotechnological applications. These include the production of compatible solutes, extracellular enzymes (adapted to saline stress), and exopolysaccharides among others.

Taxonomy, Historical and Current

Short Description of the Family

Ha.lo.mo.na.da’ce.ae. M.L. fem. n. Halomonas, type genus of the family; suff. -aceae, ending to denote a family; M.L. fem. pl. n. Halomonadaceae, the Halomonas family. The description of the family is identical to that given by Franzmann et al. (1988) and emended by Dobson and Franzmann (1996), Ntougias et al. (2007) and Ben Ali Gam et al. (2007).

The family Halomonadaceae belongs, together with the Alcanivoraceae, the Hahellaceae, the Litoricolaceae, the Oceanospirillaceae, the Oleiphilaceae and the “Saccharospirillaceae” to the order Oceanospirillales, within the class Gammaproteobacteria (Garrity et al. 2005a) that consists mainly of marine species.

The family Halomonadaceae was originally proposed by Franzmann et al. (1988) and it was later emended by Dobson and Franzmann (1996), Ntougias et al. (2007) and Ben Ali Gam et al. (2007). At the start of 2014, this family included ten recognized genera: Halomonas (type genus), Aidingimonas, Carnimonas, Chromohalobacter, Cobetia, Halotalea, Kushneria, Modicisalibacter, Salinicola, and Zymobacter (Parte 2014). Table 17.1 contains relevant taxonomic information on these genera and their species.

Table 17.1 Validly published genera and species names of the family Halomonadaceae (as to 31 January 2014). Basonyms/synonyms of microorganisms that have been transferred to other genera are not included. For genera/species whose descriptions have been emended only the most recent reference is included

As mentioned before, some of the current species were isolated and described many years before the proposal of the genera Halomonas (Vreeland et al. 1980), Chromohalobacter (Ventosa et al. 1989) or Cobetia (Arahal et al. 2002a): Pseudomonas beijerinckii (basonym of Chromohalobacter beijerinckii), “Chromobacterium marismortui” (now Chromohalobacter marismortui), “Arthrobacter marinus” (earlier synonym of Cobetia marina), “Achromobacter aquamarinus” (Halomonas aquamarina), Flavobacterium halmophilum (basonym of Halomonas halmophila) and “Micrococcus halodenitrificans” (Halomonas halodenitrificans) are the oldest examples. In 1972, Baumann and coworkers published an extensive taxonomic study of Gram-negative, nonfermentative marine bacteria, including four organisms assigned at that time to the genus Alcaligenes, namely Alc. aestus, Alc. cupidus, Alc. pacificus and Alc. venustus (Baumann et al. 1972). About one decade later, Baumann et al. (1983) proposed the creation of the genus Deleya to accommodate those four marine species as well as Pseudomonas marina.

The genera Halomonas and Deleya served as the basis for the creation of the family Halomonadaceae (Franzmann et al. 1988). At that time these genera contained four and six species, respectively. A chemotaxonomic study (Franzmann and Tindall 1990) of members of the family Halomonadaceae concluded that on the basis of respiratory quinone, polar lipid, and fatty acid compositions, no clear distinction existed at the genus level. Additionally, it was concluded that Alcaligenes aquamarinus (currently Halomonas aquamarina) and Halovibrio variabilis (Halomonas variabilis) were members of the family Halomonadaceae and could perhaps be accomodated within existing genera of the family.

Only a few months earlier Ventosa et al. (1989) proposed Chromohalobacter as a new genus with a single species, C. marismortui, on the basis of a subculture of “Chromobacterium marismortui,” isolated from the Dead Sea (Elazari-Volcani 1940), and seven moderately halophilic isolates from a Mediterranean saltern in Spain that were found to be very closely related to it. Later, in the phylogenetic study of Mellado et al. (1995b) it was concluded that this genus belongs to the family Halomonadaceae.

The genus Zymobacter, with its single species Z. palmae, was created by Okamoto et al. (1993) and placed later in the family Halomonadaceae (Dobson and Franzmann 1996).

By then, 16S rRNA phylogenetic analyses were used as definitive evidence of the lack of correlation in the taxonomic arrangements within the family Halomonadaceae (Dobson et al. 1993). Mellado et al. (1995b) proposed the reclassification of Volcaniella eurihalina as Halomonas eurihalina and pointed out the heterogeneity of the Halomonas-Deleya complex. Dobson and Franzmann (1996) transferred all species of the genus Deleya to the genus Halomonas together with Halovibrio variabilis and Paracoccus halodenitrificans. In one way, this simplification stopped the confusion of the naming within the Halomonas-Deleya complex, but the resulting genus, Halomonas, contained (and still does) very different species and it is considered too heterogeneous. The genus Halomonas was expanded to 15 species, with few characters in common, while the only two other genera recognized at that time, Chromohalobacter and Zymobacter, contained one each. Meanwhile, the genus Carnimonas was created by Garriga et al. (1998) to accommodate one single species, C. nigrificans isolated from cured meat products, and later it was included into the family Halomonadaceae (Arahal et al. 2002c). The same year, the genus Cobetia was created by Arahal et al. (2002a) to accommodate the species Halomonas marina.

More recently, other five additional genera belonging to this family have been described: Halotalea (Ntougias et al. 2007), Modicisalibacter (Ben Ali Gam et al. 2007), Kushneria (Sánchez-Porro et al. 2009), Aidingimonas (Wang et al. 2009), and Salinicola (Anan’ina et al. 2007). Besides, the genera Halomonas, Chromohalobacter, and Cobetia were expanded to include new species since new descriptions were carried out. Moreover, several reclassifications have taken place among genera of the family Halomonadaceae: the species Halomonas canadensis and H. israelensis (Arahal et al. 2001a) were transferred to the genus Chromohalobacter; the species H. avicenniae, H. indalinina, and H. marisflavi to the genus Kushneria (Sánchez-Porro et al. 2009); and the species H. salaria and Chromohalobacter salarius to the genus Salinicola (de la Haba et al. 2010b). In 2013, Romanenko et al. (2013) classified H. halodurans as a later heterotypic synonym of Cobetia marina, invalidating the former species name.

As of January 2014 there are 106 validly published species names within the family Halomonadaceae, 79 belonging to the genus Halomonas, eight to Chromohalobacter, five to Cobetia, five to Kushneria, four to Salinicola, and the genera Aidingimonas, Carnimonas, Halotalea, Modicisalibacter, and Zymobacter with a single species each one. The list of articles in press of the International Journal of Systematic and Evolutionary Microbiology (http://ijs.sgmjournals.org/content/early/recent) includes the description of a new Halomonas species for which the name H. huangheensis has been proposed.

A valuable effort to address the taxonomy of the group from an entirely phenotypic point of view was the study of Mata et al. (2002). In that article, they presented a detailed phenotypic characterization of the type strains of all Halomonas species recognized at that time and the intraspecific variation of four of those species by studying 87 additional strains. The authors compared the reactions of 234 morphological, physiological, biochemical, nutritional and antimicrobial susceptibility tests. Part of the nutritional characterization was obtained by using a miniaturized (Biolog) identification system. It was the first time that such a method was employed so extensively among halomonads. In addition to the new data that were presented in their paper, some differences were observed between their results and those from the original species descriptions. Numerical analyses demonstrated the phenotypic heterogeneity of the Halomonas species (Mata et al. 2002). An important conclusion of the study of Mata et al. (2002) is that phenotypic traits can be selected according to their usefulness for distinguishing Halomonas species.

In 2007, the International Committee on Systematics of Prokaryotes–Subcommittee on the Taxonomy of the Halomonadaceae, following Recommendation 30b of the Bacteriological Code (1990 Revision) (Lapage et al. 1992), published the minimal standards for describing new taxa within this family (Arahal et al. 2007). This paper evaluates many different approaches to ensure that a rich polyphasic characterization is given and must be considered as guidelines for authors to prepare descriptions of novel taxa. Although a list of traits that are required and recommended is provided, the manuscript does not attempt to limit the characterization of new isolates to the features that are indicated in the text. Moreover, current or yet to be described species may show new and interesting characteristics not listed in the paper of Arahal et al. (2007) and such features may prove to be of taxonomic importance. More recently, the Subcommittee on the Taxonomy of Halomonadaceae, in an open meeting held in Storrs, Connecticut, USA (June 2013), agreed that the current minimal standards document are still adequate in combination with Notes on the characterization of prokaryote strains for taxonomic purposes (Tindall et al. 2010), but the following additions were suggested: (i) It was stressed that it is highly desirable to base the description of new species on more than one strain. (ii) A good database of sequences of suitable genes for multilocus sequence analysis is now available (de la Haba et al. 2012), and inclusion of multilocus sequence analysis in the minimal standards is highly desirable. (iii) Fatty acid analysis should be added as required rather than recommended in the recommended standards for describing new taxa of the family Halomonadaceae (Oren and Ventosa 2013).

Phylogenetic Structure of the Family and Its Genera

Seven main phylogenetic studies have been conducted on the Halomonadaceae. In the first one (Franzmann et al. 1988), which was the basis for the proposal of this family, the method employed was the 16S rRNA oligonucleotide cataloguing technique. Later, Dobson et al. (1993) obtained the 16S rRNA sequences of Deleya aquamarina (now Halomonas aquamarina), Deleya halophila (Halomonas halophila), Deleya marina (Cobetia marina), Halomonas elongata, Halomonas meridiana, Halovibrio variabilis (Halomonas variabilis) and Halomonas subglaciescola and analyzed them together with the sequences of Halomonas halmophila and other species belonging to the Gammaproteobacteria. They showed that the level of 16S rRNA sequence similarity among members of the family Halomonadaceae is 100–92.6 % and that the phylogenetic grouping did not correspond to the taxonomic assignment of the species analyzed, suggesting the unification into a single genus. They also proposed a number of characteristic sequence signatures of the members of the family Halomonadaceae that have been readapted in other studies (Dobson and Franzmann 1996; Arahal et al. 2002c; Ntougias et al. 2007; Ben Ali Gam et al. 2007), as new members have been described. Currently, the 16S rRNA sequence signature characteristics of the family Halomonadaceae are defined by the following positions: 484 (A or G), 486 (C or U), 640 (A or G), 660 (A), 668 (A), 669 (A), 737 (U), 738 (U), 745 (U), 776 (U), 1124 (U or G), 1297 (U), 1298 (C), 1423 (A), 1424 (C or U), 1439 (U or C), 1462 (A or G) and 1464 (C or U). However, fullsequence analyses are much more informative than signatures alone since the latter have to be redefined on the basis of present and future new species.

Mellado et al. (1995b) conducted a phylogenetic study on six new 16S rRNA sequences corresponding to Chromohalobacter marismortui (four strains), Volcaniella eurihalina (now Halomonas eurihalina), Deleya salina (Halomonas salina), and close relatives. They proposed the reclassification of Volcaniella eurihalina as Halomonas eurihalina but highlighted the need of a polyphasic approach to determine the natural taxonomic position of members of the Halomonadaceae, especially for the genus Halomonas since its heterogeneity was (and still is) too large for a single genus.

Further studies by Dobson and Franzmann (1996) determined another seven 16S rRNA sequences corresponding to the type strains of Halomonas subglaciescola, Deleya cupida (currently Halomonas cupida), Deleya pacifica (Halomonas pacifica), Deleya salina (Halomonas salina), Deleya venusta (Halomonas venusta), Halomonas halodurans (Cobetia marina), and Halomonas eurihalina. On the basis of their results, they proposed the unification of the genera Deleya, Halomonas and Halovibrio and the species Paracoccus halodenitrificans into the genus Halomonas.

Arahal et al. (2002c) evaluated the phylogenetic status of the family Halomonadaceae using 16S and 23S rRNA gene sequences. In addition to the new sequences determined in their study, 18 for the 23S rRNA and 7 for the 16S rRNA, the sequences were compared to more than 16,000 full or almost full rRNA sequences. By that time, the number of those sequences that could be ascribed to the family Halomonadaceae exceeded 70 (including many sequences from environmental clones and poorly characterized isolates). In addition, several treeing methods were used to elucidate the most stable branchings. A good agreement between the 16S rRNA- and the 23S rRNA-based trees was obtained. According to this study, the genus Halomonas was formed by two well-defined phylogenetic groups (containing five and seven species, respectively) as well as six species that could not be assigned to any of the above-mentioned groups. Group 1 comprised Halomonas elongata (type species of the genus), Halomonas eurihalina, Halomonas halmophila, Halomonas halophila, and Halomonas salina, all bearing a 98.2 % average sequence (16S rRNA or 23S rRNA) similarity. Group 2 included the species Halomonas aquamarina, Halomonas meridiana, Halomonas magadiensis, Halomonas variabilis, Halomonas venusta, Halomonas halodurans (now Cobetia marina), and Halomonas subglaciescola, and exhibited a 97.6 % mean 23S rRNA sequence similarity (97.4 % in the case of the 16S rRNA sequences). The species Halomonas pacifica, Halomonas halodenitrificans, Halomonas cupida, Halomonas desiderata, Halomonas campisalis, and Halomonas pantelleriensis, not only did not clearly fall into either of the two groups mentioned above but also shared relatively low values of sequence similarity with them or even between themselves (91.7–96.7 %; Arahal et al. 2002c). With respect to the genus Chromohalobacter, the four species described in those days within this genus formed a group closely related to Halomonas. The average rRNA sequence similarity of species of Chromohalobacter was 98.6 % (for the 23S rRNA) and 98.5 % (for the 16S rRNA). Within this group fell the sequence of Pseudomonas beijerinckii, which was later reclassified as Chromohalobacter beijerinckii (Peçonek et al. 2006). When the Chromohalobacter sequences were compared to those of other halomonads, values below 95 % (generally accepted as a good borderline for genus separation) were obtained in all cases. Similar low values were obtained for the sequence of Halomonas marina, which forms a deeper branch of the Halomonas-Chromohalobacter group. Indeed, according to this and other data, this organism was proposed as the type species of the new genus Cobetia (Arahal et al. 2002a). Finally, in this study, the sequences of Zymobacter palmae and Carnimonas nigrificans showed a deeper branching in the tree. Their 16S rRNA sequence similarity was 93.5 % and even lower values were obtained when comparing any of the two with the other members of the family (Arahal et al. 2002c).

More recently, de la Haba et al. (2010a) updated the comparative analysis based on 23S and 16S rRNA gene sequences of Arahal et al. (2002c) including the 49 novel species that had been described since 2002. A total of 28 new complete 23S rRNA sequences were obtained in this study. Additionally, following the recommended minimal standards for the description of new members of the family Halomonadaceae, seven already-sequenced 16S rRNA genes of type strains were resequenced to resolve undetermined positions and to reach the established quality standards. In that sense, some suggestions were included in the paper about the recommended sequences to be used for future comparative phylogenetic analysis. In general, there was excellent agreement between the phylogenies based on both rRNA genes, but the 23S rRNA gene showed higher resolution in the differentiation of species of the family Halomonadaceae due to the slower evolutionary rate for the 16S rRNA gene. As previously reported by Arahal et al. (2002c), the genus Halomonas resulted to be not monophyletic and comprised two clearly separated phylogenetic groups that now contained larger numbers of species. Group 1, representing Halomonas sensu stricto, was formed by Halomonas elongata (the type species of the genus), H. eurihalina, H. caseinilytica, H. halmophila, H. sabkhae, H. almeriensis, H. halophila, H. salina, H. organivorans, H. koreensis, H. maura and H. nitroreducens. The mean 16S rRNA gene sequence similarity for this group was 97.8 %, whereas a lower value was obtained with the 23S rRNA gene sequences (97.0 %). Group 2, included the 16 species Halomonas aquamarina, H. meridiana, H. axialensis, H. magadiensis, H. hydrothermalis, H. alkaliphila, H. venusta, H. boliviensis, H. neptunia, H. variabilis, H. sulfidaeris, H. subterranea, H. janggokensis, H. gomseomensis, H. arcis and H. subglaciescola. This group displays mean similarities of 97.4 % and 97.5 % for the 16S and 23S rRNA gene sequences, respectively. Similarity values between groups 1 and 2 were low enough as to suggest that they could constitute two different genera, however, neither chemotaxonomic nor more general phenotypic studies have permitted their separation. The other 27 species at that time assigned to the genus Halomonas did not appear to be included clearly in either of these phylogenetic groups. One of these species, H. salaria, formed a separate cluster with the species Chromohalobacter salarius and Salinicola socius, which was confirmed by further studies proposing the transference of the first two species to the genus Salinicola (de la Haba et al. 2010b). An important finding in the paper of de la Haba et al. (2010a) is the fact that the type species of Halomonas halodurans and Cobetia marina shared 100 % sequence similarity (16S and 23S rRNA) and, according to their data, there was not sufficient evidence to determine whether they were members of the same or different species. A recent publication has demonstrated that, actually, they belong to the same genospecies (Romanenko et al. 2013). Concerning the genus Chromohalobacter, all the species described until then clustered together (the mean 16S and 23S rRNA gene sequence similarity of this group was 98.0 % and 97.8 %, respectively) with the only exception being Chromohalobacter salarius, as discussed previously (de la Haba et al. 2010a). Finally, the phylogenetic distinctness of the remaining genera at that time included in the family Halomonadaceae (Carnimonas, Cobetia, Halotalea, Kushneria, Modicisalibacter, Salinicola, and Zymobacter) was confirmed in the mentioned paper, being stable in the trees produced from all methods of analysis.

In the meanwhile, 26 new species have been proposed within the genus Halomonas, some of them belonging to the group 1 (H. beimenensis, H. sinaiensis, H. smyrnensis, and H. stenophila), others to the group 2 (H. alkaliantarctica, H. andesensis, H. cibimaris, H. hamiltonii, H. jeotgali, H. johnsoniae, H. nanhaiensis, H. olivaria, H. stevensii, H. titanicae, H. vilamensis, and H. zhanjiangensis) and others that cannot be assigned to any of these two groups (H. daqiaonensis, H. flava, H. fontilapidosi, H. ilicicola, H. qijiaojingensis, H. ramblicola, H. rifensis, H. xianhensis, H. xinjiangensis, and H. zincidurans) (Fig. 17.1 ). Besides, four novel species of the genus Cobetia, and one of the genera Kushneria and Salinicola, respectively, have been described, all of them forming a monophyletic branch with the other relatives of their respective genera (Fig. 17.1 ). Additionally, the new genus Aidingimonas, not included in the study of de la Haba et al. (2010a), clustered together with the genus Modicisalibacter (Fig. 17.1 ), but they share ≤95 % 16S rRNA similarity values among them and with respect to the other genera of the family Halomonadaceae.

Fig. 17.1

Phylogenetic reconstruction of the family Halomonadaceae based on 16S rRNA and created using the neighbour-joining algorithm with the Jukes-Cantor correction. The sequence datasets and alignments were used according to the All-Species Living Tree Project (LTP) database (Yarza et al. 2010; http://www.arb-silva.de/projects/living-tree). 1,000 resampling boostrap values over 50 % are shown. Scale bar indicates estimated sequence divergence

From the phylogenetic point of view, some well-defined relationships can be observed within members of the Halomonadaceae. These groups, as defined above, are stable regardless of the methodology employed. Other relations may become better defined once more in-between sequences become available.

The most recent phylogenetic study conducted within the family Halomonadaceae was carried out by de la Haba et al. (2012). A multilocus sequence analysis (MLSA) of 52 representative species was performed for the first time in a moderately halophilic bacterial group with the purpose of investigating in detail the phylogenetic relationships of species from the family Halomonadaceae and helping clarify the current classification of this complex and dynamic family. A total of six loci were selected for the analysis, the 16S and 23S rRNA and the following four protein-encoding genes (housekeeping genes): atpA (F1-ATP synthase, α subunit), gyrB (DNA gyrase, B subunit), rpoD (RNA polymerase, β subunit) and secA (protein translocase, SecA subunit). Different nucleotide substitution models and tree-constructing algorithms were compared. The average pairwise sequence similarity values for 16S rRNA, 23S rRNA, atpA, gyrB, rpoD and secA were 94.0 %, 93.1 %, 86.8 %, 79.7 %, 79.6 % and 76.6 %, respectively, indicating that the secA gene had the highest theoretical discriminatory power, although some halomonads secA gene sequences were identical, suggesting gene flow. In any case, the six genes studied were not always sufficient to correctly assign a new strain to the genus Halomonas since there was a large overlap between the intrageneric and intergeneric sequence similiarities, that is, halomonads sequences could be more similar to those from species belonging to other genera of the family than to those from species of the genus Halomonas. This overlap was mainly due to the enormous variability within the genus Halomonas, suggesting that the genus should be divided into two or more genera. Besides, the overlap problem also lies with the huge sequence similarity of the pair Halomonas halodurans-Cobetia marina, whose taxonomic status has been recently revised by Romanenko et al. (2013) and concluded that they are member of the same species. With respect to the phylogenetic trees, the different methods produced variable results, with those generated from the maximum-likelihood and neighbour-joining algorithms being more similar than those obtained by maximum-parsimony methods. Except atpA gene, the other five genes studied showed a consistent evolutionary history (with some exceptions probably due to lateral gene transfer events, and other intrinsic and extrinsic factors, such as the size of the dataset and the taxa included); therefore atpA gene may not be useful as an individual gene phylogenetic marker within the family Halomonadaceae. The gyrB-based tree was the only that formed monophyletic branches for the different genera of the family, including the genus Halomonas. Although there were some exceptions, in general, the two groups defined within this genus by Arahal et al. (2002c) and de la Haba et al. (2010a) (group 1 or Halomonas sensu stricto and group 2) could be clearly distinguished in the phylogenetic trees for each gene. Concatenation of the six loci enhanced the phylogenetic reconstruction and optimized the taxonomic resolution by adding more informative data and minimizing the weight of recombination events. Trees resulting from the six-gene concatenation demonstrated a monophyletic and well-supported separation of the different genera, including the genus Halomonas. The only exceptions were the pairs Halomonas halodurans-Cobetia marina (mentioned above, with concatenated sequence similarity of 99.7 %) and Halomonas muralis–M. tunisiensis (with concatenated sequence similarity of 90.8 %). With regard to the intrageneric groups, the six-gene concatenations resolved Halomonas groups 1 and 2 monophyletically, demonstrating to be a very good tool for the delineation of taxonomic relationships on a broad scale, including intrageneric and intergeneric relationships, at the level of the family Halomonadaceae. In order to simplify the MLSA approach within this family, de la Haba et al. (2012) attempted to reduce the number of genes to be analyzed but retaining the resolution obtained with the six concatenated gene sequences. With this idea the general use of the individual and concatenated 16S rRNA, gyrB and rpoD genes is suggested for future taxonomic studies using MLSA within the family Halomonadaceae. This proposal has been recently endorsed and recommended within the minimal standards by the ICSP-Subcommittee on the Taxonomy of the Halomonadaceaae (Oren and Ventosa 2013). Finally, the paper of de la Haba et al. (2012) analyze the phylogeny of this family within the domain Bacteria by comparing the sequences obtained in this study with those of 445 bacterial species with sequenced genomes available from the GenBank/EMBL/DDBJ databases. The results showed that the family Halomonadaceae constituted a robust and monophyletic branch within the domain Bacteria for three of the six analysed genes, 16S rRNA, gyrB and rpoD. Besides, according to Figs. 17.1, 17.2 , and 17.3 the family is related to families Saccharospirillaceae, Litoricolaceae, Oceanospirillaceae, and Hahellaceae.

Fig. 17.2

Phylogenetic distribution of the genus Halomonas based on 16S rRNA and created using the neighbor-joining algorithm with the Jukes-Cantor correction. The sequence datasets and alignments were used according to the All-Species Living Tree Project (LTP) database (Yarza et al. 2010; http://www.arb-silva.de/projects/living-tree). 1,000 resampling boostrap values over 50 % are shown. Scale bar indicates estimated sequence divergence

Fig. 17.3

Phylogenetic distribution of genera within the family Halomonadaceae other than Halomonas based on 16S rRNA and created using the neighbor-joining algorithm with the Jukes-Cantor correction. The sequence datasets and alignments were usedaccording to the All-Species Living Tree Project (LTP) database (Yarza et al. 2010; http://www.arb-silva.de/projects/living-tree). 1,000 resampling boostrap values over 50 % are shown. Scale bar indicates estimated sequence divergence

Molecular Analyses

DNA-DNA Hybridization Studies

DNA–DNA hybridization data between type strains of species contained in the family Halomonadaceae are widely available. Actually, the vast majority of descriptions include results of DNA-DNA hybridization (DDH). As indicated in the recomended minimal standards for this family, DDH studies remains essential when novel species are described (Arahal et al. 2007), and only in those proposals based on a single isolate that possess less than 97 % 16S rRNA gene sequence similarity with its closest relative can DDH data be considered redundant.

With respect to DDH between strains belonging to the same species within the family Halomonadaceae data are very limited, mainly due to the fact that the majority of the species have been described based on a single isolate.

Multilocus Sequence Analysis (MLSA)

With the objective to overcome the limitations attached to DNA-DNA hybridization studies (time-consuming, expensive, lack of uniformity and reproducibility problems, etc.) and to 16S rRNA gene sequence based taxonomy (high levels of conservation, microheterogeneity among the different copies, lateral gene transfer, etc.), additional rRNA and protein-encoding genes (called housekeeping genes) have been suggested as phylogenetic markers (Stackebrandt et al. 2002; Zeigler 2003; Arahal et al. 2007; Tindall et al. 2010) to perform multilocus sequence analysis (MLSA).

Although some authors (Arahal et al. 2002c; Lee et al. 2005; de la Haba et al. 2010a; Okamoto et al. 2004; González-Domenech et al. 2010) had previously used some housekeeping genes (23S rRNA, gyrB, ectBC, narH, nirS, and nosZ) for studying the family Halomonadaceae, the first MLSA study was conducted by de la Haba et al. (2012) based on six phylogenetic markers: 16S rRNA, 23S rRNA, atpA, gyrB, rpoD, and secA genes. However, the correlation of DDH values against the MLSA data could not be determined and, therefore, this approach cannot be taken yet as an alternative to DDH for species circumscription.

DNA Fingerprinting Methods

Determination of inter- and intraspecies relatedness by rapid DNA typing methods (AFLP, RAPD, rep-PCR, BOX-PCR, PFGE, ribotyping of ribosomal ribonucleic operons, ARDRA) has not been widely applied to the Halomonadaceae except for the studies by Mellado et al. (1998) and Llamas et al. (2002), who applied PFGE, Garriga et al. (1998), who used RAPD, Heyrman et al. (2002), who reported rep-PCR, and Li et al. (2008) who carried out BOX-PCR genomic fingerprinting.

Genes Sequenced and Characterized

Several genes from different members of the family Halomonadaceae have been sequenced and characterized, mainly from Halomonas elongata (Göller et al. 1998; Grammann et al. 2002; Kraegeloh et al. 2005; Schwibbert et al. 2011) and Chromohalobacter salexigens (Cánovas et al. 1998, 2000; Copeland et al. 2011), but also from Cobetia marina (Kraiwattanapong et al. 1999), Halomonas eurihalina (Llamas et al. 2003), Halomonas halodenitrificans (Sakurai and Sakurai 1998; Sakurai et al. 2006), Halomonas maura (Llamas et al. 2006), Halomonas meridiana (Coronado et al. 2000b), Halomonas organivorans (Moreno et al. 2011), Halomonas salina (Sripo et al. 2002), and Zymobacter palmae (Raj et al. 2002).

Genome Sizes and Plasmid

Before the genomic era, estimations of the genome size of 11 Halomonas and Chromohalobacter strains were carried out by using pulsed-field gel electrophoresis (Mellado et al. 1998; Llamas et al. 2002; Quesada et al. 2004). Additionally, the presence of plasmids (and megaplasmids) in strains of Halomonas, Chromohalobacter and Cobetia has been investigated (Fernández-Castillo et al. 1992; Vargas et al. 1995; Mellado et al. 1995a; Llamas et al. 1997; Argandoña et al. 2003).

Genome Comparison

As of January 2014, complete or draft genome sequences were available for type strains of the following species: Carnimonas nigrificans (JAGO00000000), Chromohalobacter salexigens (CP000285), Halomonas anticariensis (ASTJ00000000, AUAB00000000), Halomonas boliviensis (AGQZ00000000), Halomonas elongata (FN869568), Halomonas halocynthiae (AUDZ00000000), Halomonas jeotgali (AMQY00000000), Halomonas lutea (ARKK00000000), Halomonas smyrnensis (AJKS00000000), Halomonas stevensii (AJTS00000000), Halomonas titanicae (AOPO00000000), Halomonas zhanjiangensis (ARIT00000000), and Kushneria aurantia (ARNK00000000). Although they have not been sequenced yet, the type species of Halomonas halodenitrificans, Halotalea alkalilenta, and Zymobacter palmae are part of the Genomic Encyclopedia of Type Strains, Phase I: the 1,000 microbial genomes (KMG) project.

Additional sequenced strains are: Halomonas sp. 23_GOM-1509m (JADJ01000000), Halomonas sp. A3H3 (CBRE000000000), Halomonas sp. BJGMM-B45 (AVBC00000000), Halomonas sp. GFAJ-1 (AHBC00000000), Halomonas sp. HAL1 (AGIB00000000), Halomonas sp. HTNK1 (Gi05412), Halomonas sp. KM-1 (BAEU00000000), and Halomonas sp. TD01 (AFQW00000000).

A summary of the genome characteristics of the sequenced species of the family Halomonadaceae is presented in Table 17.2 .

Table 17.2 Genome characteristics of some members of the family Halomonadaceae

Phenotypic Analyses

Halomonadaceae Franzmann et al. (1989), emend. Dobson and Franzmann (1996), Ntougias et al. (2007), and Ben Ali Gam et al. (2007)

Ha.lo.mo.na.da’ce.ae. M.L. fem. n. Halomonas, type genus of the family; suff. -aceae, ending to denote a family; M.L. fem. pl. n. Halomonadaceae, the Halomonas family.

The members of the family Halomonadaceae cannot be defined by a reasonably large number of common-to-all features. This phenotypic heterogeneity of the family is also a handicap for the identification at the genus or species level unless a sufficient number of characters are determined. Cells are Gram-negative, straight or curved, rod-shaped. They are either slight or moderate halophiles or halotolerant, except species of the genus Zymobacter (Okamoto et al. 1993), growing in the presence of high concentrations of sugars. Aerobic or facultatively anaerobic, chemoorganotrophs (Dobson and Franzmann 1996; Garrity et al. 2005b). Mata et al. (2002) reviewed in depth the phenotypic features of the genus Halomonas, and confirmed the enormous diversity among the species of this genus. In addition, they reported a large number of traits not analyzed previously for all strains and found tests that are useful for distinguishing the species of the genus Halomonas.

Genotypic diversity within the Halomonadaceae is also huge, as indicated for the genomic DNA G+C content, which ranges from 52.0 to 74.3 mol% (Martínez-Cánovas et al. 2004b; Arahal and Ventosa 2006).

From the chemotaxonomic point of view, the fatty acid profile is available for most species described within this family whereas the analysis of respiratory lipoquinones or polar lipids has been addressed for only some of them. There are a set of features shared for the vast majority of the species belonging to the Halomonadaceae. The major respiratory lipoquinone is ubiquinone 9 (Q9), although ubiquinone 8 (Q8) and ubiquinone 10 (Q10) are also present in several species. With regards to polar lipid composition all the members possess phosphatidylethanolamine and phosphatidylglycerol (Franzmann and Tindall 1990), except for the species Aidingimonas halophila, which contains diphosphatidylglycerol instead of phosphatidylglycerol (Wang et al. 2009). The fatty acid profile notably varies depending on the growing media and conditions, but in general, the main fatty acids are C_16:0, C_18:1ω7c, C_16:1ω7c, C_19:0 cyclo ω8c, C_17:0 cyclo, and C_12:0 3-OH.

Table 17.3 shows the phenotypic and chemotaxonomic features, as well as the DNA G+C content comparison among the genera of the family Halomonadaceae. The type genus of the family is Halomonas (Vreeland et al. 1980).

Table 17.3 Phenotypic and chemotaxonomic characteristics and DNA G+C content of genera of the family Halomonadaceae

Halomonas Vreeland et al. (1980), emend. Dobson and Franzmann (1996)

Ha.lo.mo’nas. Gr. n. hals, halos salt of the sea; Gr. n. monas a unit, monad; M.L. fem. n. Halomonas salt(-tolerant) monad.

Gram-negative, straight or curved, rod-shaped cells, generally 0.6–0.8 × 1.6–1.9 μm, except the species H. halodenitrificans that presents coccoid cells. Some species may produce poly-β-hydroxyalcanoates and/or exopolysaccharides. Endospores are not formed. Motile by means of peritrichous, lateral or polar flagella or nonmotile. Colonies are white to yellow, turning light brown with age. Slight to moderate halophiles, that are able to grow in NaCl concentrations ranging from 0.1 % to 32.5 % (w/v). Possess a mainly respiratory type of metabolism with oxygen as the terminal electron acceptor, but some species are also capable of anaerobic growth in the presence of nitrate. Some species have been reported to grow under anaerobic conditions in the absence of nitrate if supplied with glucose (but not other carbohydrates or amino acids). Some species reduce nitrate to nitrite; nitrogen gas is not formed. Catalase positive and most of them are also oxidase positive. Chemoorganotrophic. Carbohydrates, organic acids, polyols, and amino acids, can be used as sole carbon and energy sources or as sole carbon, nitrogen and energy sources (Vreeland et al. 1980; Dobson and Franzmann 1996; Vreeland 2005).

The major respiratory lipoquinone is ubiquinone 9 (Vreeland 2005), with the exception of H. alkaliphila that mainly possess ubiquinone 8 and ubiquinone 6 (Romano et al. 2006). The major fatty acids are C_16:1 ω7c, C_17:0 cyclo, C_16:0, C_18:1 ω7c, and C_19:0 cyclo ω8c (Vreeland 2005). The predominant polar lipids are phosphatidylethanolamine and phosphatidylglycerol.

DNA G+C content ranges between 52.0 and 74.3 mol% (Martínez-Cánovas et al. 2004b; Arahal and Ventosa 2006), demonstrating the enormous heterogeneity within this genus.

Halomonas is the type genus of the family Halomonadaceae. The species Halomonas elongata is the type species of the genus.

Aidingimonas Wang et al. (2009)

Ai.ding.i.mo’nas. N.L. n. Aiding a lake located in Xinjiang province of north-west China; L. fem. n. monas, monad a unit, a monad; N.L. fem. n. Aidingimonas a monad from Aiding Lake.

Cells are Gram-negative, facultatively anaerobic, non-endospore-forming, short rods. Non-motile, without flagella. Moderately halophilic. Positive for catalase activity. Negative for oxidase activity and nitrate reduction. Ubiquinone 9 is present. Major fatty acids are C_19:0 cyclo ω8c and C_16 : 0. The polar lipid pattern consists of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides, two unknown phospholipids, two unknown phosphoglycolipids and one unknown glycolipid. The DNA G+C content is 57.2–57.5 mol% (Wang et al. 2009).

The only species within this genus is Aidingimonas halophila, which was isolated from a salt lake in Xinjiang province, north-west China. Cell size ranges between 0.1–0.3 × 0.7–1.5 μm. It forms colourless to yellow brown colonies, flat and opaque with slightly irregular edges. Growth occurs at 10–45 °C, at pH 5.0–10.0 and in 1–25 % (w/v) NaCl, with optimal growth at 37 °C, pH 7.0–8.0 and 5–10 % NaCl. Does not contain poly-β-hydroxybutyrate granules or produce exopolysaccharide. Growth occurs under anoxic conditions in the presence of nitrate ion as electron acceptor. The Voges–Proskauer test is variable. Indole and H₂S are not produced. Milk peptonization and coagulation and the methyl red test are negative. Gelatin, aesculin, casein, starch, and Tweens 40, 60 and 80 are not hydrolysed, but positive for hydrolysis of Tween 20 and urea. ONPG, phenylalanine deaminase and lysine and ornithine decarboxylase tests are negative, but positive for arginine dihydrolase. Citrate and other substrates can be utilized as a sole carbon or nitrogen and energy sources. Acid is produced from different carbohydrates and organic acids. The type strain is YIM 90637^T, with a DNA G+C content of 57.5 mol% (Wang et al. 2009).

Carnimonas Garriga et al. (1998)

Car.ni’mo.nas. L. gen. n. carnis of meat; Gr. n. monas a unit, monad. Carnimonas a monad of meat.

Straight or slightly curved rods, 0.5–0.6 × 1.0–1.7 μm, occurring singly or in pairs. Gram-negative. Does not form endospores. Nonmotile. Oxidase and catalase positive. Aerobic, having a strictly respiratory type of metabolism with oxygen as the terminal electron acceptor. Slightly or moderate halophile. No growth occurs in the presence of more than 8 % (w/v) NaCl. Optimum temperature for growth is 28–30 °C. No growth occurs at 5 °C or 37 °C. Chemoorganotrophic. Acid, but no gas, is produced from d-glucose, d-xylose, melibiose, maltose and sucrose. β-galactosidase (ONPG) activity occurs. Forms dark spots on the surface of raw, cured meat products. The main respiratory quinone is ubiquinone-9. Main components in the polar lipid composition are diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. Major fatty acids are C_16:0, C_16:1, C_18:1, and C_19:0 cyclo. The G+C content of the DNA is 56.0 mol% (Garriga et al. 1998; 2005).

The type and the single species within this genus is Carnimonas nigrificans. In the species description a total of nine strains, CTCBS1^T to CTCBS9 were reported to be isolated from cured meat products. Colonies are non-pigmented, white, convex, shiny and circular. Aesculin and starch are hydrolysed. Gelatin, casein and DNA are not hydrolysed. Voges-Proskauer negative. Arginine dihydrolase, urease, lecithinase and phenylalanine deaminase negative. Indole is not produced. Nitrate is not reduced (Garriga et al. 1998; 2005).

Chromohalobacter Ventosa et al. (1989) emend. Arahal et al. (2001a)

Chro.mo.ha’lo.bac’ter. Gr. n. chroma color; Gr. n. halos the sea, salt; M.L. n. bacter rod; M.L. masc. n.Chromohalobacter colored salt rod.

Gram-negative, straight or sometimes slightly curved, rods (0.4–1.2 × 0.8–6.1 μm). Motile by polar or peritrichous flagella. Cells occur singly, in pairs, and in short chains. Colonies are cream to brown-yellow pigmented, with the exception of C. nigrandesensis that shows black pigmentation. Endospores are not formed. Moderately halophilic. Salt is required for growth. The optimum salt concentration for growth is between 8 % and 10 %. May grow at salt concentrations up to 30 %. The broader ranges of temperature and pH observed for growth are 0–45 °C (optimal 30–37 °C) and pH 5.0–10.0 (optimal pH 7.5), respectively. Aerobic. Chemoorganotrophic. Catalase positive. Oxidase negative, with the exception of C. beijerinckii (Peçonek et al. 2006) and C. sarecensis (Quillaguamán et al. 2004a). Some strains reduce nitrates, but H₂S and urease are not produced, with the exception of C. nigrandesensis (Prado et al. 2006) and C. salexigens (Arahal et al. 2001b). Phenylalanine deaminase test is negative. Starch, Tween 80, aesculin, DNA, and tyrosine are not hydrolyzed. The species C. japonicus is the only able to hydrolyse gelatin (Sánchez-Porro et al. 2007). Acid is produced aerobically from d-glucose and other carbohydrates. Carbohydrates, amino acids, and some polyols can serve as sole carbon or nitrogen sources (Arahal et al. 2001a; Ventosa 2005).

Predominant fatty acids are C_16:0, C_19:0 cyclo ω8c, and C_18:1 ω7c; C_17:0 cyclo, and C_12:0 3-OH are present in smaller amounts. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and two unknown phospholipids are major to moderate compounds in the polar lipid profile. The quinone system consists of the major compound Q-9 (>95 %) and small amounts of Q-8 (Peçonek et al. 2006; Sánchez-Porro et al. 2007). The DNA G+C base composition ranges from 56.1 to 66.0 mol%.

The type species of the genus is Chromohalobacter marismortui, previously named as “Chromobacterium marismortui”, which was originally isolated from the Dead Sea (Ventosa et al. 1989).

Cobetia Arahal et al. (2002b) emend. Romanenko et al. (2013)

Co.be’ti.a. N.L. fem. n. Cobetia named after A. B. Cobet, who originally described the type species as “Arthrobacter marinus”.

Gram-negative, straight, rod-shaped cells that are 1.6–4.0 × 0.8–1.2 μm, and occur singly and in pairs. Strains are non-motile or motile by means of a single polar flagellum and/or two to seven lateral flagella. Some strains can produce fimbria-like structures and capsules. Colonies are round, bright, smooth and cream pigmented. Poly-β-hydroxyalkanoate is accumulated. Oxidase negative. Aerobic; unable to grow anaerobically in the presence of nitrate or arginine. Sodium ions are not essential for growth. Most strains can grow without addition of NaCl to the medium, but optimal growth occurs in the presence of 5–6 % (w/v) salts and they can considered as slightly halophiles. Good growth is also obtained up to 20 % (w/v) but not at higher salinities. Hydrolyses tyrosine, but not gelatine, starch or Tween 80. Hydrolysis of casein, DNA and aesculin is strain-dependent (negative reaction for most strains). Negative for chitin hydrolysis and Simmons’ citrate test. ONPG positive. Phosphatase positive. Negative for phenylalanine deaminase, methyl red, indole production, and nitrate reduction. H₂S is not produced. Acid is produced from glucose and other carbohydrates (Arahal et al. 2002a; Romanenko et al. 2013).

Polar lipids include phosphatidylethanolamine, phosphatidylglycerol, unknown phospholipids, unknown lipids, an unknown aminolipid and phosphatidic acid. The major fatty acids are C_16:1 ω7c, C_16:0, C_12:0 3-OH, C_18:1 ω7c and C_17:0 cyclo. The DNA G+C content varies between 61.4 and to 64.2 mol% (Kim et al. 2010b; Romanenko et al. 2013).

The species Cobetia marina is the type of the genus and strain DSM 4741^T is the type strain of this species. Strain DSM 5160, formerly the type strain of the species Halomonas halodurans, has been proposed as member of C. marina based on phylogenetic analysis and DNA-DNA hybridization and chemotaxonomic characteristics (Romanenko et al. 2013).

Halotalea Ntougias et al. (2007)

Ha.lo.ta.le’a. Gr. n. hals halos salt; L. fem. n. talea a staff, rod; N.L. fem. n. Halotalea rod-shaped cells living in saline conditions.

Cells are Gram-negative, rods, motile by peritrichous flagella and forming small, non-pigmented pale yellow colonies. Endospores are not formed. Strictly aerobic. Halotolerant and alkalitolerant. Sugar-tolerant. Oxidase- and catalase-positive. Chemo-organotrophic. Ubiquinone-9 is present in the respiratory chain. The major fatty acids are C_18:1 ω7c, C_16:0, C_19:0 cyclo ω8c, C_12:0 3-OH and C_16:1 ω7c/iso-C_15:0 2-OH. The DNA G+C content is 64.4 mol% (Ntougias et al. 2007).

The type species is Halotalea alkalilenta, which tolerates up to 15 % (w/v) NaCl, with an optimum salt concentration of 0–3 % (w/v) NaCl. Tolerates up to 45 % and 60 % w/v (+)-d-glucose and maltose, respectively. Grows at pH 5–11, with optimum at pH 7. The temperature range for growth is 5–45 °C, with an optimum temperature of 32–37 °C. Negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, phenylalanine deaminase, nitrate reduction, H₂S production from l-cysteine, indole production, methyl red and Voges-Proskauer. Acid is produced from (+)-d-glucose and other carbohydrates. Hydrolyses Tween 20, but does not hydrolyse casein, DNA, gelatin, starch, Tween 80 or urea. The type strain is AW-7^T, isolated from olive mill waste (alkaline alpeorujo) obtained from the premises of the Toplou Monastery in the region of Sitia, Crete, Greece (Ntougias et al. 2007).

Kushneria Sánchez-Porro et al. (2009)

Kush.ne’ri.a. N.L. fem. n. Kushneria from the name Kushner, honouring Dr Donn J. Kushner, a Canadian microbiologist who carried out pioneering studies on halophilic micro-organisms.

Cells are Gram-negative, motile rods (0.5–2.0 × 1.7–5.0 μm). Endospores are not formed. Strictly aerobic. Colonies are yellow-orange to cream. Moderately halophilic; Na⁺ is required for growth. The optimal NaCl range supporting the growth is 0.5–12 % (w/v). Mesophilic (able to grow from 4 to 42° C, optimum at 25–37° C). Growth occurs at pH 4.5–10.0 (optimally at pH 7.0–8.0). Chemo-organotrophic. Catalase-positive and oxidase-negative. Aesculin and gelatin are hydrolysed, with the exception of Kushneria sinocarnis that does not possess gelatinase activity (Zou and Wang 2010). Casein, starch, Tween 80, DNA, and tyrosine are not hydrolysed. Indole and H₂S production, and Voges-Proskauer test are negative. Phosphatase is produced, but urease, arginine dihydrolase, and lysine decarboxylase are not. Except K. indalinina (Cabrera et al. 2007) and K. sinocarnis (Zou and Wang 2010), the other species do not reduce nitrate to nitrite. The major respiratory quinone is Q9. Major fatty acids are C_16:0, C_18:1 ω7c, C_19:0 cyclo ω8c, C_12:0 3-OH, and C_17:0 cyclo. Polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and unidentified phospholipids and glycolipids. The DNA G+C content is 59.0–61.7 mol% (Yoon et al. 2001; Cabrera et al. 2007; Soto-Ramírez et al. 2007; Sánchez-Porro et al. 2009; Zou and Wang 2010).

The type species of the genus is Kushneria aurantia, with the type strain A10^T isolated from the leaf surface of Avicennia germinans (black mangrove).

Modicisalibacter Ben Ali Gam et al. (2007)

Mo’di.ci.sa’li.bac’ter. L. adj. modicus moderate, limited; L. n. sal, salis salt; N.L. masc. n. bacter a rod; N.L. masc. n. Modicisalibacter a moderately halophilic rod.

Cells are Gram-negative, non-endospore-forming, motile rods. Moderately halophilic. Strictly aerobic and require Na⁺ for growth. Mesophilic, growing well at 15–45 °C, oxidase-negative and reduce nitrate. Predominant fatty acids are C_16:0, C_18:1 ω7c, C_16:1 ω7c, C_19:0 cyclo ω8c and C_17:0. Contents of C_19:0 cyclo ω8c and C_17:0 differ significantly from those of other members of the Halomonadaceae. The DNA G+C content is 53.7 mol% (Ben Ali Gam et al. 2007).

The genus contains a single species, namely Modicisalibacter tunisiensis, whose cells are approximately 1.0–4.0 μm long × 0.6–1.0 μm wide. Colonies on marine agar are circular, smooth, convex and 2–3 mm in diameter after 48 h of incubation at 37 °C. Cells grow at 4–45 °C, with optimum at 37 °C. The pH range for growth is 5–10, with an optimum at pH 7.2. Growth occurs in the range 0.1–25 % (w/v) NaCl and optimally at 10 % (w/v) NaCl. Catalase reaction is positive. ONPG hydrolysis is negative. Citrate is not utilized. Urease and arginine dihydrolase are not produced. Gelatin, alginate and aesculin are not hydrolysed. H₂S and indole are not produced. d-glucose, d-fructose, tryptone, peptone and Casamino acids are utilized. The type strain is LIT2^T, which was isolated from a sample of oilfield-water injection collected in the Sidi Litayem area near Sfax, Tunisia (Ben Ali Gam et al. 2007).

Salinicola Anan’ina et al. (2008)

Sa.li.ni.co’.la. L. fem. pl. n. salinae salterns, salt-works; N.L. suff. -cola, derived from incola, inhabitant; N.L. masc. n. Salinicola, inhabitant of salterns.

Cells are Gram-negative, non-endospore-forming rods (0.5–1.3 × 1.0–3.2 μm). Motile by means of a single lateral/polar flagellum or by peritrichous flagella (Huo et al. 2013). Colonies are circular, smooth, convex and creamy yellow-coloured. Aerobic and chemoorganotrophic. Moderately halophilic that grows in the range of 0–30 % (w/v) NaCl with the optimum of 0.5–20 % (w/v) NaCl, at pH 4.5–10.0 (optimum pH 5.0–8.0). Mesophilic, growing in the range 4–45 °C (optimal growth temperature is 25–37 °C). Exopolysaccharides are not produced, but the species Salinicola salarius and S. socius produce poly-β-hydroxyalkanoate (de la Haba et al. 2010b). Respiration on fumarate, nitrate and nitrite is negative. Shows positive reaction in the oxidation/fermentation of d-glucose. Catalase reaction is positive, oxidase reaction is negative, except for S. salarius (Kim et al. 2007). Tween 20 is hydrolysed. Tyrosine and aesculin are not hydrolysed. β-Galactosidase is not produced. Phosphatase activity is present. For all species, except S. peritrichatus, methyl red test is positive and Voges-Proskauer is negative (de la Haba et al. 2010b; Huo et al. 2013). Indole is not produced. Lysine- and ornithine-decarboxylases and phenylalanine deaminase are negative. Gluconate is not oxidized. Selenite is not reduced. The predominant respiratory lipoquinone is ubiquinone with nine isoprene units (Q9). The major fatty acids are C_16:1 ω7c, C_16:0, C_18:1 ω7c, C_19:0 cyclo ω8c, and C_12:0 3-OH. The DNA G+C content ranges between 58.8 and 63.6 mol% (Anan’ina et al. 2007; Kim et al. 2007; Aguilera et al. 2007; de la Haba et al. 2010b; Huo et al. 2013).

The type species of the genus is Salinicola socius, isolated from the microbial community obtained from the soil of salt mines (Berezniki, Perm region of Russia), which grows on naphthalene as a sole carbon and energy source. The other three species comprising the genera are S. halophilus (formerly Chromohalobacter salarius), S. peritrichatus, and S. salarius (basonym of Halomonas salaria). The species name S. zeshunii (Cao et al. 2013) has been effectively, but not validly published.

Zymobacter Okamoto et al. (1995)

Zy.mo.bac’ter. Gr. n. zyme leaven, ferment; M.L. n. bacter masc. equivalent of Gr. neut. n. bakterion rod; M. L. masc. n. Zymobacter the fermenting rod.

Non-endospore forming rod-shaped cells with rounded ends, 1.3–2.4 × 0.7–0.9 μm; usually single. Motile by as many as 20 peritrichous flagella that are non-sheathed. Gram-negative. Facultatively anaerobic. Chemoorganotrophic. Grow on and ferment 1 mol of glucose or hexose moiety of maltose to produce approximately 2 mol each of ethanol and CO₂, with a trace amount of acids. Ferments hexoses, α-linked di- and tri-saccharides and sugar alcohols. Growth initiates at pH values of 4.7–8.1. Catalase-positive and oxidase-negative. The major cellular fatty acids are C_16:0, C_19:0 cyclo, C_18:1 ω9c, and C_12:0 3-OH. Quinone system is ubiquinone-9. The DNA G+C base composition ranges from 55.4 to 56.2 mol% (Okamoto et al. 1993, 2005).

Currently, the genus include a single species, Zymobacter palmae, with the type strain T109^T isolated from palm sap. Colonies are round, entire, smooth, opaque, and milky white. Colonies of similar size develop under aerobic and anaerobic growth conditions. Growth is better in static cultures than in shaken cultures. Requires nicotinic acid for growth. Growth does not occur in the absence of a sugar or sugar alcohol. Growth occurs at 15–37 °C (optimum, 30 °C) and pH 4.7–8.1 (optimum, pH 6.0). The organisms are neither halophilic nor halotolerant, but they are considerably tolerant to ethanol and produce ethanol mainly from maltose (5.8 % ethanol after 6 days of fermentation), but also from glucose, fructose, sucrose, melibiose, raffinose, sorbitol, mannitol, and –depending on the strain– mannose and galactose. Methyl red, Voges-Proskauer and α-glucosidase are positive. The following tests are negative: indole production, utilization of citrate, nitrate reduction, chromogenicity, gelatin liquefaction, hydrolysis of starch, phenylalanine deaminase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and β-galactosidase (Okamoto et al. 1993, 2005).

Isolation and Maintenance Procedures

Isolation

Halomonads may be isolated following standard microbiological techniques on complex defined media with a suitable salinity and incubated at room temperature for 1–7 days.

Since not all species show the same requirements (or tolerance) of salinity (Table 17.4 ), differences are expected to occur depending on the final salt content of the media. With the only exception of Zymobacter palmae, all species are able to grow at salinities in the range of 5–10 % but not necessarily at their optimum over the whole range. Media for the selective isolation of moderately halophilic bacteria can be prepared with higher salt contents (for instance 20 %) to prevent the growth of non-adapted competitors. The selectivity of such media can be increased by lowering the concentration of Mg²⁺ to prevent the growth of extremely halophilic archaea (Ventosa et al. 1982). However, not all the species within the family Halomonadaceae are able to grow at such high salinities.

Table 17.4 Physiological features of member of the family Halomonadaceae

The growth of species of Aidingimonas, Chromohalobacter, some Halomonas (such as H. alkaliantarctica, H. alkaliphila, H. almeriensis, H. anticariensis, H. aquamarina, H. beimenensis, H. campaniensis, H. cerina, H. cibimaris, H. cupida, H. daqiaonensis, H. daqingensis, H. denitrificans, H. eurihalina, H. fontilapidosi, H. gomseomensis, H. gudaonensis, H. halmophila, H. halodenitrificans, H. halophila, H. ilicicola, H. janggokensis, H. jeotgali, H. korlensis, H. lutea, H. maura, H. olivaria, H. organivorans, H. pantelleriensis, H. sabkhae, H. shengliensis, H. sinaiensis, H. smyrnensis, H. stenophila, H. subglaciescola, H. taeanensis, H. variabilis, H. ventosae, H. vilamensis, and H. xinjiangensis), and the species Kushneria aurantia, K. indalinina, K. sinocarnis, Modicisalibacter tunisiensis, Salinicola halophilus, and S. salarius will be favored in media with a salt content of 7.5–10 %. Some of them will yield reasonable growth at 15 % salts or even at 20 %. At these concentrations, growth of extremely halophilic Archaea can occur, but they are easily distinguished by their red pigmentation. Most of the above-mentioned species have a minimum requirement for NaCl of 0.5–5 %.

Other species (Cobetia amphilecti, Cob. crustatorum, Cob. litoralis, Cob. marina, Cob. pacifica, Halomonas alimentaria, H. boliviensis, H. campisalis, H. elongata, H. flava, H. hamiltonii, H. hydrothermalis, H. koreensis, H. kribbensis, H. mongoliensis, H. muralis, H. nitroreducens, H. qijiaojingensis, H. ramblicola, H. rifensis, H. saccharevitans, H. salina, H. stevensii, H. xianhensis, H. zincidurans, K. avicenniae, K. marisflavi, and Salinicola socius) are also moderately halophilic, but their optimum salinity for growth is lower (around 5 %). Most of these species show no requirement of salts or require only as little as 0.5–1 %.

The slightly halophilic species (Halomonas andesensis, H. arcis, H. axialensis, H. caseinilytica, H. desiderata, H. halocynthiae, H. johnsoniae, H. kenyensis, H. magadiensis, H. meridiana, H. nanhaiensis, H. neptunia, H. pacifica, H. salifodinae, H. subterranea, H. sulfidaeris, H. titanicae, H. venusta, H. zhanjiangensis, Halotalea alkalilenta and S. peritrichatus) grow optimally at around 3 % NaCl. However, these species are halotolerant of up to 15–20 % NaCl (25 % in the case of H. neptunia and H. titanicae and 10 % in the case of H. kenyensis).

Carnimonas nigrificans shows only a limited level of halotolerance. For its isolation, two selective media have been proposed (Garriga et al. 1998), cetrimide agar and MacConkey agar, although it may grow in other media such as tryptone soy agar. Although the organism is not pigmented, it produces black spots on the surface of cured meat products (the isolation source). This coloration seems to be the consequence of nonenzymatic reactions of substances derived from the meat surface.

On the contrary, Zymobacter palmae is neither halophilic nor halotolerant, but other special features of this organism can be used for its isolation. Thus, Okamoto et al. (1993) first selected ethanol-tolerant bacteria using media with 5 % (v/v) ethanol and then tested isolates for production of ethanol from maltose. Zymobacter palmae was found to produce ethanol from the fermentation of a variety of sugars: hexoses, α-linked di- and tri-saccharides, and sugar alcohols (fructose, galactose, glucose, mannose, maltose, melibiose, saccharose, raffinose, mannitol and sorbitol).

Regarding pH, most species are neutrophilic and therefore the pH of media can be adjusted to around 7.2–7.5. Exceptions to this rule are H. alkaliantarctica, H. alkaliphila, H. campaniensis, H. campisalis, H. daqingensis, H. desiderata, H. kenyensis, H. korlensis, H. magadiensis, H. mongoliensis, and H. pantelleriensis that grow optimally at pH 9.0 or 9.5 or Cobetia crustatorum, Salinicola peritrichatus, and Zymobacter palmae, with optimal growth at pH 5 or 6 (Table 17.4 ).

Finally, although all halomonads are mesophilic, some differences in their behavior towards temperature can be used for selective isolation. Aidingimonas halophila, Chromohalobacter japonicus, C. marismortui, C. salexigens, Cobetia amphilecti, C. litoralis, C. marina, C. pacifica, Halomonas alkaliphila, H. beimenensis, H. campaniensis, H. desiderata, H. elongata, H. flava, H. fontilapidosi, H. halophila, H. ilicicola, H. kenyensis, H. lutea, H. magadiensis, H. meridiana, H. mongoliensis, H. organivorans, H. qijiaojingensis, H. ramblicola, H. rifensis, H. sabkhae, H. smyrnensis, H. titanicae, H. xinjiangensis, Halotalea alkalilenta, Kushneria aurantia, K. sinocarnis, Modicisalibacter tunisiensis, Salinicola peritrichatus, and S. socius grew well at 37 °C while the rest grew only suboptimally (or not at all). Temperatures around 30 °C fit all known species so far. In the lower limit most species show no apparent growth below 4–15 °C (20 °C in the case of H. anticariensis, H. flava, H. magadiensis, H. qijiaojingensis, Kushneria aurantia, and Z. palmae; 25 °C in the case of H. ilicicola, H. rifensis, and H. sinaiensis; 30 °C in the case of H. sabkhae), but Chromohalobacter sarecensis, H. boliviensis, H. subglaciescola are capable of growing at 0 °C, and H. axialensis, H. neptunia, H. sulfidaeris at −1 °C. With respect to the upper limit, the species H. alkaliphila, H. beimenensis, H. campisalis, H. daqingensis, H. denitrificans, H. mongoliensis, H. olivaria, H. sabkhae, H. sinaiensis, H. ventosae, and H. xinjiangensis are able to grow up to 50 °C, although the most thermotolerant species is H. kenyensis, growing up to 55 °C (Table 17.4 ).

Maintenance and Preservation

A variety of media have been described for the routine maintenance of halomonads strains in the laboratory. In many cases the same media served also for the isolation of the strains, whereas in others different formulations were employed. Some of these formulations, for instance the Artificial Organic Lake (AOL) medium of Franzmann et al. (1987), are intended to mimic the chemical composition of the environment from where the organisms are isolated. Once that isolation has been achieved many authors find it advantageous, especially if preparation of the isolation medium is too laborious, to employ a more general medium that permits the growth of both fresh isolates and reference strains. Thus, one of the most commonly employed media is MH, for moderately halophilic bacteria (Ventosa et al. 1982).

MH medium (Ventosa et al. 1982)
Yeast extract	10 g
Proteose peptone	5 g
Glucose	1 g
NaCl	81 g
MgCl2 · 6H2O	7 g
MgSO4 · 7H2O	9.6 g
CaCl2 · 2H2O	0.36 g
KCl	2 g
NaHCO3	0.06 g
NaBr	0.026 g
Distilled water q.s.	1 L

Adjust pH to 7.2 with 1 M KOH or NaOH. Add agar (20 g liter⁻¹) for preparation of solid media. Adjust total saline content (commonly 10 %) to any other desired value by lowering or raising proportionally the amounts of salts.

In 2002 Mata et al. employed MH medium at 7.5 % salt content to maintain 104 Halomonas strains (including 21 type strains). For the vast majority of the strains the pH was adjusted to 7.2–7.5, except for two of the strains, H. magadiensis NCIMB 13595^T and H. campisalis ATCC 700597^T, for which the pH was adjusted to 9. All the Halomonas species described from 2002 up to now can be mantained in the same medium at pH 7.2–7.5, with the exception of H. alkaliantarctica, H. alkaliphila, H. campaniensis, H. daqingensis, H. desiderata, H. kenyensis, H. korlensis, H. mongoliensis, and H. pantelleriensis that grow optimally at pH 9.0 or 9.5.

For the species of the family Halomonadaceae with a lower salt requirement, commercial media such as Marine Agar (MA) can be satisfactorily employed (Yoon et al. 2001; Heyrman et al. 2002; Romanenko et al. 2002, 2013; Kim et al. 2010b). Alternatively, other commercial media such as trypticase soy agar (TSA) can also be employed by adding NaCl (or a mixture of salts) or even no salts (for the non-halophilic species) (Garriga et al. 1998). With regards to the species Zymobacter palmae (non-halophile) the MY medium is recommended (Okamoto et al. 1993), which consisted of 1 % Bacto yeast extract (Difco), 2 % maltose, 0.2 % KH₂PO₄ and 0.5 % NaCl, pH 6.0. The species Cobetia crustatorum and Salinicola peritrichatus are also slightly acidophiles (with optimal growth at pH 5 or 6), but they can be mantained in MH medium at 7.5 % salts.

Cultures on agar slants can be sealed and stored at 4–10 °C. Although viability may last for much longer periods it is recommended that they be transferred regularly every 1–3 months.

For long-term preservation, lyophilization is advised. Prior to the vacuum drying, actively growing cells can be suspended on protecting fluids such as 5 % inositol solution, and then the vials can be frozen by immersion into liquid nitrogen.

Cryopreservation at −80 °C or under liquid nitrogen is also possible. To enhance survival of the cells they have to be suspended with a cryoprotectant such as 20 % glycerol solution. Such prepared vials can also be stored at −20 °C for middle-term preservation. However, since the quality of the culture may diminish faster (especially after frequent freeze-thawing) it is recommended that new stocks be prepared regularly.

Ecology

Habitat

According to the original description of the family Halomonadaceae (Franzmann et al. 1988), its members typically occurr in “temperate and Antarctic saline lakes, solar salt facilities, saline soils and marine environments”. This is still true for the majority of the current species (Table 17.5 ). Indeed the only exceptions to the above definition are Carnimonas nigrificans, Chromohalobacter beijerinckii, Chr. canadensis, Chr. japonicus, Cobetia crustatorum, Halomonas alimentaria, H. cibimaris, H. daqingensis, H. desiderata, H. halodenitrificans, H. hamiltonii, H. jeotgali, H. johnsoniae, H. muralis, H. olivaria, H. stevensii, Halotalea alkalilenta, Kushneria sinocarnis, Modicisalibacter tunisiensis, and Zymobacter palmae. Of these 20 organisms, only Zymobacter palmae is neither halophilic nor halotolerant (however, it is very tolerant to ethanol –up to 6 %).

Table 17.5 Habitats of members of the family Halomonadaceae from which the original strains were isolated

So, the halomonads can be found in any saline environment, regardless of its geographical location. This includes oceans and seas (even at considerable depths), saline soils, salty foods, naturally occurring saline lakes, solar pans, etc. Since some of its members are also alkaliphilic, they are found in soda lakes and alkaline soils. Additionally, three species have been isolated from blood patients and from dialysis machines of a renal care centre.

On the genus level, not surprisingly, Halomonas is found to be the most ubiquitous genus with the largest number of species, and these are very heterogeneous.

As for the interactions of halomonads with other microorganims, Ivanova et al. (2002) characterized a heterotrophic microbial enrichment community established during the degradation of brown algae Fucus evanescens, and consisting of two species, Pseudoalteromonas sp. and C. marina. While the first was highly metabolically active (14 hydrolytic activities could be detected) and likely plays the main role in the initial stages of algal degradation, the second, C. marina, produced only caseinase and DNase but was resistant to the bacteriolytic activity of the former and utilized the degradation products of polysaccharides.

In a study about the temporal stability and biodiversity of two complex antilisterial cheese-ripening microbial consortia (Maoz et al. 2003), out of 400 isolates, three were identified as H. venusta, two as H. variabilis, and two as Halomonas sp. by Fourier-transform infrared spectroscopy and 16S ribosomal RNA sequence analysis.

A recent study based on the metagenomic analysis of two hypersaline saltern ponds (19 % and 37 % NaCl) from Santa Pola (Spain) has demonstrated that the genera Halomonas and Chromohalobacter, which are commonly obtained in pure culture from similar salinity samples, are almost not represented in those ponds according to the metagenomic reads obtained (Ghai et al. 2011). This means that members of Halomonadaceae are less abundant in hypersaline environment than it was previously thought when using culture-dependent techniques.

The diversity and distribution of Halomonas populations in the hypersaline habitat Rambla Salada, situated in south-east Spain, have been studied using different molecular techniques (Oueriaghli et al. 2014). Denaturing gradient gel electrophoresis (DGGE) using specific primers for the 16S rRNA gene of Halomonas followed by a multivariate analysis of the results indicated that richness and evenness of the Halomonas populations were mainly influenced by the season, being the summer (the season with the highest salinity) the one with the highest value of diversity. Furthermore, canonical correspondence analysis (CCA) demonstrated that both salinity and pH significantly affected the structure of the Halomonas community. Halomonas almeriensis and two denitrifiers, H. ilicicola and H. ventosae were the predominant species. CARD-FISH showed that the percentage of Halomonas cells with respect to the total number of microorganisms in that habitat ranged from 4.4 % to 5.7 %. Finally, no significant differences between the types of samples studied, from either watery sediments or soil samples, were found (Oueriaghli et al. 2014). Classical cultivation methods have been also employed very recently to analyze the diversity of the halophilic bacterial community from Rambla Salada, being Halomonas the most abundant genus, representing 41.2 % of the 364 isolated strains (Luque, R., Béjar, V., Quesada, E., and Llamas, I., unpublished).

Pathogenicity, Clinical Relevance

Members of the Halomonadaceae were thought to be not pathogenic. There is one case report on the isolation of H. venusta from a human infection in a wound that originated from a fish bite (Von Graevenitz et al. 2000); however the identification of the organism alone does not prove its pathogenicity.

In 2007, Berger et al. (2007) reported an outbreak of “Halomonas phocaeensis” bacteraemia in a neonatal intensive care unit in Tunisia, attributed to contamination from a water bath used to warm fresh frozen plasma.

In a study performed seeking bacterial DNA signatures in unexplained deaths and critical illnesses, polymerase chain reaction of 16S RNA gene from culture-negative materials identified an unspeciated Halomonas organism in one patient’s blood (Nikkari et al. 2002). The phylogenetic analysis of the sequence data determined that this organism was part of the H. variabilis–H. boliviensis–H. neptunia cluster, but in an apparently distinct position (Stevens et al. 2009). Additionally, Stevens et al. (2009) isolated a total of 14 strains recognized as human pathogens causing infection and contamination in a dyalisis center. Exhaustive taxonomic characterization of these isolates led to the description of three new Halomonas species, H. stevensii, H. hamiltonii and H. johnsoniae (Kim et al. 2010a).

More recently, a patient developed a bacteremia caused by Halomonas johnsoniae (previously reported only as dialysis unit environmental contaminants) (Stevens et al. 2013). The medical community is alerted to the pathogenic potential of the genus in humans, but also in algae and animals (Kim et al. 2013).

Applications

The species of the Halomonadaceae can be used for several biotechnological purposes and, as in the case of other extremophilic microorganisms, many different applications have been suggested. The biotechnological potential and applications of moderately and halotolerant microorganisms have been reviewed in detail (Ventosa et al. 1998; Margesin and Schinner 2001; Mellado and Ventosa 2003; Quillaguamán et al. 2010; Oren 2010). Some of the most promising applications of members of Halomonadaceae include the production of compatible solutes and polyhydroxyalkanoates as well as extracellular compounds such as exopolysaccharides and enzymes, and their use in environmental bioremediation processes.

Compatible solutes are known for their stabilizing and protective effect on enzymes, nucleic acids, cell structures or whole cells subjected to low water activities, temperature stress, and other adverse conditions (Lippert and Galinski 1992; Galinski 1993; Knapp et al. 1999) and therefore could be useful for industrial and clinical purposes. A “bacterial milking” process to obtain ectoine from Halomonas elongata has been developed and patented (Sauer and Galinski 1998) and later industrially exploited by Bitop (Witten, Germany). The process is based on subjecting the bacteria repeatedly to osmotic shocks. An osmotic down-shock permits the excretion of the intracellular ectoine to the surrounding medium while subsequent exposure of the cells to a hyperosmotic shock quickly restores the original level of ectoine (Sauer and Galinski 1998). Very recently, an ectD (ectoine hydroxylase) deficient H. elongata mutant has been proved to produce ectoine from a variety of sugars derived from lignocellulosic biomass and thus has tremendous potential as a host for producing useful compounds from biomass resources (Tanimura et al. 2013). Concerning other halomonads, a process comprising two-step fed-batch cultivation has been investigated for the production of ectoine and hydroxyectoine using Halomonas boliviensis DSM 15516^T (Guzmán et al. 2009; Van-Thuoc et al. 2010a, b). The first cultivation was performed under optimal conditions for cell growth and resulted in a high cell mass concentration. During the second cultivation at higher salt concentration, accumulation of ectoines increased while cell mass decreased. Maximum productivity of total ectoines reached was 10 g l⁻¹ d⁻¹ at 18.5 % NaCl, which is among the highest reported so far. The accumulated ectoines were released by subjecting the cells to hypoosmotic shock and the cells were further recycled for the production process (Van-Thuoc et al. 2010a). A similar method in two stages has been employed for efficient ectoine production using Halomonas salina DSM 5928^T (Zhang et al. 2009; Lang et al. 2011). An ectoine absorption defective H. salina DSM 5928^T mutant (lacking the ectoine-specific transporter TeaABC), which compromised the negative feedback regulation of ectoine synthesis, was constructed to improve the efficiency of ectoine production up to 9.93 g l⁻¹ d⁻¹ (Xu and Zhang 2012). Apart from Halomonas species, only the species Chromohalobacter salexigens has been optimized for ectoine and hydroxyectoine production (Fallet et al. 2010; Rodríguez-Moya et al. 2013). Ectoine and its derivative hydroxyectoine are used in the cosmetic industry because of their moisturizing properties. The potential use of the Chromohalobacter salexigens and Halomonas elongata ect genes (responsible for the synthesis of ectoine) to obtain agriculturally important transgenic organisms tolerant to osmotic stress has already been proposed (Vargas et al. 2004). Nakayama et al. (2000) obtained transgenic cultured tobacco cells that accumulated the compatible solute ectoine from H. elongata, exhibiting a normal growth pattern under hyperosmotic conditions.

Poly-β-hydroxyalkanoate (PHA) is a polymer accumulated by many prokaryotes, that can be used for the production of biodegradable plastics (“biological polyesters”) with properties resembling that of polypropylene. Several bacterial species of the family Halomonadaceae accumulate PHAs: Cobetia amphilecti, Cob. crustatorum, Cob. litoralis, Cob. marina, Cob. pacifica, Chromohalobacter salexigens, Chr. sarecensis, Halomonas almeriensis, H. andesensis, H. anticariensis, H. alkaliphila, H. aquamarina, H. boliviensis, H. campaniensis, H. campisalis, H. caseinilytica, H. cerina, H. cibimaris, H. cupida, H. daqiaonensis, H. daqingensis, H. desiderata, H. elongata, H. eurihalina, H. fontilapidosi, H. halmophila, H. halodenitrificans, H. halophila, H. hamiltonii, H. jeotgali, H. johnsoniae, H. magadiensis, H. maura, H. meridiana, H. nitroreducens, H. olivaria, H. pacifica, H. pantelleriensis, H. ramblicola, H. rifensis, H. salina, H. sinaiensis, H. stevensii, H. subglaciescola, H. variabilis, H. ventosae, H. venusta, H. zhanjiangensis, Kushneria marisflavi, Salinicola salarius, and S. socius. From those, the strain Halomonas boliviensis LC1^T reached PHA yields and volumetric productivities close to the highest reported so far, accumulating the compound to up to 88 % of its dry weight (Quillaguamán et al. 2006, 2007). Furthermore, H. boliviensis and other Halomonas species are able to co-produce PHA and osmolytes, i.e., ectoines and hydroxyectoine, in one process (Quillaguamán et al. 2010).

Strains from several species of Halomonas are of interest as producers of exopolysaccharides (EPS). These include H. alkaliantarctica, H. alkaliphila, H. almeriensis, H. anticariensis, H. caseinilytica, H. cerina, H. daqiaonensis, H. daqingensis, H. eurihalina, H. fontilapidosi, H. halophila, H. maura, H. nitroreducens, H. olivaria, H. ramblicola, H. rifensis, H. sabkhae, H. salina, H. sinaiensis, H. smyrnensis, H. stenophila, and H. ventosae. EPS production has also been described in the species Cobetia crustatorum. The nutritional and environmental factors influencing the production of EPS have been thoroughly investigated (Quesada et al. 1993; Béjar et al. 1996; Béjar et al. 1998; Bouchotroch et al. 1999; Bouchotroch et al. 2000; Martinez-Checa et al. 2002; 2007; Arias et al. 2003; Mata et al. 2006; Llamas et al. 2012). Yield can reach 1–3 g of EPS per liter of medium after five days of cultivation. Glucose and sucrose are the most efficient carbon sources (Béjar et al. 1996) although many other nutrients can be used including end products such as molasses from sugar beet (Quesada et al. 2004). Production of the EPS is not inhibited by the presence of crude oil. Moreover, the composition of the biopolymer is different under such conditions showing a highly efficient emulsifying activity towards crude oil (Calvo et al. 2002). Interestingly, it has been observed that strains of H. maura and H. eurihalina produce more EPS at salt concentrations below 7.5 % (w/v), which are suboptimal for growth (Quesada et al. 1993; Bouchotroch et al. 2000). As for the chemical composition of the EPSs produced from Halomonas strains, they are anionic polymers composed mainly of carbohydrates and a minor fraction of proteins, uronic acids and acetyls. Sulfate substituents have also been detected (in some strains exceeding 20 % of the dry weight), and together with the uronic acids, make the EPS anionic. Depending on their composition EPSs have different functional properties and thus different applications, which include immunomodulation, biodetoxification of heavy-metal polluted environments, crude oil emulsification, viscosity enhancement in foods, protection against oxidative stress, among others (Quesada et al. 2004; Raveendran et al. 2013). Recently, it has been demonstrated that the EPS produced by H. stenophila strain B100 selectively induces apoptosis in human T leukaemia cells (Ruiz-Ruiz et al. 2011), suggesting that the search for new antineoplastic drugs should include the screening of other bacterial EPSs, particularly those isolated from halophiles. The properties of some of these EPSs such as mauran (from H. maura), H28 and V2-7 (from H. eurihalina) and those from strains Al12^T and Al16 (H. ventosae), from strains FP35^T and FP36 (H. anticariensis) and from strain M8^T (H. almeriensis) have been studied in detail highlighting in each case their biotechnological potentials (Pérez-Fernandez et al. 2000; Martinez-Checa et al. 2002, 2007; Arias et al. 2003; Mata et al. 2006; Llamas et al. 2012). Besides, genes involved in the biosynthesis of the exopolysaccharide mauran produced by H. maura have been identified, which form part of a gene cluster epsABCDJ (Arco et al. 2005). Furthermore, production of EPS in H. anticariensis was found to be influenced by a two-component regulatory system, GacS/GacA (Tahrioui et al. 2013b).

Another interesting field is the production of extracellular enzymes (i.e., amylases, proteases and nucleases) by moderately halophilic representatives of the family. Although few studies have been carried out, this is a promising subject that has been reviewed (Mellado et al. 2004). Sanchez-Porro et al. (2003) studied the diversity of moderately halophilic bacteria able to produce several extracellular hydrolytic enzymes (amylases, DNases, lipases, proteases and pullulanases) by screening different hypersaline locations in South Spain. In contrast to the scarce hydrolytic activity shown by culture collection strains, environmental isolates produce a variety of extracellular enzymes that could be of potential biotechnological interest. Identification at the genus level revealed that 25 strains and 2 out of 122 isolates belonged to Halomonas and Chromohalobacter, respectively. Another study with the aims to isolate and characterize the cultivable community of hydrolase producers inhabiting heavy-metal-contaminated soils in extreme conditions from the Atacama Desert showed that only 1 out of 25 isolates was closely related to the genus Halomonas, in particular to H. organivorans, possessing protease, lipase, amylase, DNase, and pullulanase activities (Moreno et al. 2012). Concerning the characterization of extracellular enzymes, an α-amylase produced by Halomonas meridiana has been studied at the biochemical and molecular level (Coronado et al. 2000a, b). The amylase showed optimal activity at 10 % NaCl, 37 °C and pH 7.0 (being relatively stable under alkaline conditions). The enzyme showed activity at high salt concentrations (up to 30 %). The main products resulting from the hydrolysis of starch were maltose and maltotriose. The gene encoding this α-amylase (amyH) has been cloned and expressed in the heterologous hosts H. elongata and the non-halophilic bacterium E. coli (Coronado et al. 2000b). It encodes a 457-residue protein with a molecular mass of 50 kDa. Besides, an extracellular α-amylase gene from the hyperthermophilic archaeon Pyrococcus woesei has been cloned and expressed in H. elongata, under the control of a native H. elongata promoter (Frillingos et al. 2000). More recently, a novel endoglucanase (Cel8H) from Halomonas sp. strain S66-4 has been biochemically characterized, cloned, expressed in E. coli and purified. The purified recombinant enzyme had an optimal activity of 4.9 U/mg at pH 5 and 45° C toward the substrate carboxymethylcellulose. It exhibited extraordinary properties which differed from endoglucanases reported previously at the point of high salt tolerance above 5 M, simultaneously with high stability at pH 4–12 and 40–60° C (Huang et al. 2010). Moreover, an α-glucosidase (HaG) with a estimated molecular mass of 58 kDa was isolated from Halomonas sp. strain H11 (Ojima et al. 2012a). HaG showed high hydrolytic activities toward maltose, sucrose, and p-nitrophenyl-α-d-glucoside but to almost no other disaccharides or malto-oligosaccharides higher than trisaccharides. HaG showed optimum activity to maltose at 30 °C and pH 6.5. Monovalent cations, such as K⁺, Rb⁺, Cs⁺, and NH₄ ⁺ increased the enzymatic activity to 2- to 9-fold of the original activity. This enzyme was tested to be useful for efficient synthesis of α-d-glucosylglycerol and α-glucosylated 6-gingerol (Ojima et al. 2012a, b).

Halomonads and other moderately halophilic bacteria can be also used for the degradation of toxic compounds both at high and intermediate salt concentrations, permitting the restoration of saline industrial residues and contaminated saline environments (Mellado and Ventosa 2003). However, few studies have been carried out. Some examples of halomonads with a potential for biodegradation and biotransformation of contaminants (such as aminomethane sulfonate, p-aminosalicylate, organophosphonates, benzoate, 3-chlorobenzoate, cinnamate, p-coumarate, crude-oil, 2,4-dichlorophenoxyacetate, ferulate, formaldehyde, p-hydroxybenzoate, phenol, phenylacetate, phenylpropionate, salicylate, selenate and uranium compounds) are shown in Table 17.6 . Maltseva et al. (1996) isolated halomonads able to use chloroaromatic compounds as the sole source of carbon and energy and studied in detail the degradation of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Kleinsteuber et al. (2001) reported the use of the alkaliphilic, moderately halophilic Halomonas sp. strain EF43 for the expression of the 2,4-d-degradative pathway by conjugation of the broad host range plasmid pJP4. This strain was able to degrade 2,4-d- and 3-chlorobenzoate under alkaline conditions in the presence of an additional carbon source. In 2004, García et al. (2004) described a novel species, Halomonas organivorans, which was able to use benzoic acid, p-hydroxybenzoic acid, cinnamic acid, salicylic acid, phenylacetic acid, phenylpropionic acid, phenol, p-coumaric acid, ferulic acid and p-aminosalicylic acid. Later, a screening of moderately halophilic bacteria able to use aromatic compounds permitted the isolation of a large number of members of the genus Halomonas able to degrade different compounds (García et al. 2005). Recently, cat and ben genes, involved in phenol and benzoate degradation, respectively, from H. organivorans, have been cloned, characterized and analyzed (Moreno et al. 2011), providing an ideal model system to investigate the potential use of this group of extremophiles in the decontamination of saline environments. Other recent publications also show the ability of H. organivorans to degradate contaminants, such as crude-oil (Hassanshahian et al. 2012) and phenol (Bonfá et al. 2013). The tolerance patterns of several Halomonas species and Chromohalobacter marismortui to ten heavy metals have been studied, as well as the influence of salinity and composition of culture media (Nieto et al. 1989). These studies may be interesting for future use of metal-tolerant halophilic strains as biological detoxicants.

Table 17.6 Organic compounds and chemicals that can be degraded or transformed by Halomonadaceae strains under hypersaline conditions

Few studies have been carried out on the role of halomonads in the fermentation processes of foods and other products. Cobetia crustatorum, Halomonas alimentaria, H. cibimaris, and H. jeotgali were isolated from the traditional Korean fermented seafood (Kim et al. 2010b; Yoon et al. 2002; Jeong et al. 2013; Kim et al. 2011). The hydrolytic activity of H. elongata in bacon curing brines has been investigated (Hinrichsen et al. 1994).

Fuel ethanol production has been proposed using the pyruvate decarboxylase (PDC) of Z. palmae as biocatalyst (Raj et al. 2002). This enzyme is thermostable and showed the highest specific activity and lowest Km for pyruvate of all four known bacterial PDCs.