Journal of Biotechnology 152 (2011) 114124

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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Review

Halophilic anaerobic fermentative bacteria

Anniina T. Kivist , Matti T. Karp
Tampere University of Technology, Department of Chemistry and Bioengineering, Tampere, Finland

a r t i c l e

i n f o

a b s t r a c t
In hypersaline environments bacteria are exposed to a high osmotic pressure caused by the surrounding high salt concentrations. Halophilic microorganisms have specic strategies for balancing the osmotic pressure and surviving in these extreme conditions. Halophilic fermentative bacteria form taxonomically and phylogenetically a coherent group mainly belonging to the order Halanaerobiales. In this review, halophilic anaerobic fermentative bacteria in terms of taxonomy and phylogeny, special characteristics, survival strategies, and potential for biotechnological applications in a wide variety of branches, such as production of hydrogen, are discussed. 2010 Elsevier B.V. All rights reserved.

Article history: Received 12 March 2010 Received in revised form 17 August 2010 Accepted 23 August 2010 Available online 9 September 2010 Keywords: Halophilic Fermentative Bacteria Thermophilic Alkaliphilic

Contents 1. 2. 3. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Taxonomy and phylogeny . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Extremophiles among halophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Survival methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potential for biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Halofermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Food biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Biopolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Biological waste treatment and biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. Alternative energy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 115 117 117 117 119 120 120 120 121 121 121 121 122 122 122 122

4. 5.

6.

1. Introduction Halophiles are microorganisms that require high salt concentration for growth. They are found among all kingdoms of life: bacteria, eukarya, and archaea. According to the salt concentration required for optimal growth, halophiles are categorized as halotolerant, moderately halophilic and extremely halophilic microorganisms. The halotolerant microorganisms can survive and grow even in rel-

Corresponding author. Tel.: +358 40 1981141; fax: +358 3 3115 2869. E-mail address: anniina.kivisto@tut. (A.T. Kivist). 0168-1656/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2010.08.014

atively high concentrations of salt but rather live in the absence of it, whereas the moderate halophiles achieve their optimal growth at salt concentrations from 0.5 M (25 g/l) to 2.5 M (150 g/l) and the extreme halophiles at salt concentrations over 2.5 M to saturation (340 g/l) (Joo and Kim, 2005; Ventosa et al., 1998). The halophilic archaeal members are typically either aerobic or methanogenic, and fermentative halophilic archaea are extremely rare. Most of the halophilic fermentative bacteria belong phylogenetically to the order Halanaerobiales (previously named as Haloanaerobiales) which is considered as the order of halophilic fermentative bacteria and which, until recently, comprised solely of halophilic fermentative bacteria. The order

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Halanaerobiales is placed in the phylum of Firmicutes and in the class of Clostridia and is further divided into two families, the Halanaerobiaceae (previously named as Haloanaerobiaceae) and the Halobacteroidaceae (Mavromatis et al., 2009; Oren, 2006). Altogether, the order Halanaerobiales currently includes 25 fermentative species including two subspecies. In addition to the Halanaerobiales, one species of obligate halophilic fermentative bacteria is belonging to the order Clostridiales, and one to the order Natranaerobiales. Halophilic microorganisms might be advantageous because hypersalinity suppresses the growth of most other organisms and therefore, sterilization costs can be reduced. Biotechnological applications of halophilic archaea include the production of bacteriorhodopsin, polymers, enzymes, and compatible solutes (Ventosa and Nieto, 1995). While much of the interest in halophilic bacteria has been focused on their enzymes, possible compatible solutes, and biodegradation of residues and wastes, fermentative bacteria might also produce useful fermentation products such as hydrogen, ethanol, and propanediol (Dan et al., 2003; Kapdan and Erten, 2007; Ventosa and Nieto, 1995). The topic has previously been reviewed as follows: halophilic bacteria by Lowe et al. (1993), Madigan and Oren (1999), Mesbah and Wiegel (2008), Ollivier et al. (1994) and Oren (2006), taxonomy by Rainey et al. (1995), phylogenetic and metabolic diversity by Oren (2002, 2008), survival strategies by Litcheld (1998), halophilic proteins by Mevarech et al. (2000), Fukuchi et al. (2003), and Joo and Kim (2005), bioenergetics in hypersaline environments by Oren (1999, 2001), and halophilic potential for biotechnology by Margesin and Schinner (2001), and Ventosa and Nieto (1995). Since the latest reviews concerning halophilic fermentative bacteria, new strains have been isolated and more information concerning the group has become available including some genetic information. The aim of this review is to discuss about halophilic anaerobic fermentative bacteria in terms of taxonomy and phylogeny, special characteristics, survival strategies, and potential for biotechnological applications of a wide variety of branches.

2. Taxonomy and phylogeny The rst halophilic fermentative bacterial species, Halanaerobium praevalens was isolated from the sediments of Great Salt Lake (Utah) and characterized in 1983 (Zeikus et al., 1983). Although H. praevalens was observed to distinguish in characteristics from the halophilic bacteria and archaea described previously, the species was placed in the family Bacteroidaceae as a genus with uncertain afliation (Zeikus et al., 1983). The characterization of H. praevalens was followed by the isolation and characterization of Halobacteroides halobius in 1984 from the sediments of Dead Sea and similarities in 16S rRNA sequences between the two halophilic fermentative bacteria were observed leading to placement of the species in a new family, named as Haloanaerobiaceae (Oren et al., 1984a,b). The family Haloanaerobiaceae obtained its third genus in 1987, when Clostridium lortetii, isolated from the sediments of Dead Sea and originally characterized in 1983, was transferred to a new genus of the family and renamed as Sporohalobacter lortetii (Oren, 1983; Oren et al., 1987). Between the years 1987 and 1995, ve new halophilic fermentative genera, Halothermothrix, Halocella, Acetohalobium, Halanaerobacter, and Orenia, were characterized and placed in the family Haloanaerobiaceae (Cayol et al., 1994b; Liaw and Mah, 1992; Oren et al., 1987; Simankova et al., 1993; Zhilina and Zavarzin, 1990). Eventually, in 1995, a novel order (named as Haloanaerobiales) for the halophilic fermentative bacteria was proposed (Rainey et al., 1995). In addition, according taxonomic studies and phylogenetic analyses of the halophilic fermentative

bacterial genera, a novel family (named as Halobacteroidaceae) besides the family Haloanaerobiaceae was proposed and the genera Halobacteroides, Acetohalobium, Halanaerobacter, Sporohalobacter, and Orenia were re-assigned to the novel family (Rainey et al., 1995). Most of the halophilic fermentative bacteria phylogenetically belong to the order Halanaerobiales which is considered as the order of halophilic fermentative eubacteria and which until recently comprised solely of halophilic fermentative bacteria. The order Halanaerobiales is placed in the phylum of Firmicutes and in the class of Clostridia and further divided into two families, the Halanaerobiaceae and the Halobacteroidaceae (Mavromatis et al., 2009; Oren, 2006). Altogether, the order Halanaerobiales currently includes 25 fermentative species including two subspecies. Recently, the order Halanaerobiales have increased with two nonfermentative species, Halanarsenatibacter silvermanii (unpublished name) which is a newly isolated and characterized member of the family Halanaerobiaceae and Selenihalobacter shriftii which is a member of the family Halobacteroidaceae (Switzer Blum et al., 2001, 2009). The non-fermentative species characterized were observed to grow by anaerobic respiration of arsenic and selenium compounds, respectively (Switzer Blum et al., 2001, 2009). Although the majority of the halophilic fermentative bacteria belong to the order Halanaerobiales, there are few exceptions: A halothermophilic fermentative bacterium Thermohalobacter berrensis was assigned to the order Clostridiales and the family Clostridiaceae, and haloalkalithermophilic bacterium Natranaerobius thermophilus to a new order Natranaerobiales and the new family Natranaerobiaceae based on characteristics and similarities in 16S ribosomal RNA sequences (Cayol et al., 2000; Mesbah et al., 2007). The taxonomic structure with order, family, genera, species, and subspecies of the halophilic fermentative bacteria is shown in Table 1. In addition, facultative halophilic and halotolerant fermentative bacteria that do not require salt for growth are found among genera Bacillus, Lactobacillus, Halobacterium, and Tetragenococcus. However, in this review the main focus is on salt-requiring, obligate halophilic fermentative bacteria. The family Halanaerobiaceae consist of species of the genus Halanaerobium, Halothermothrix, Halocella, and Halarsenatibacter (unpublished name) whereas the family Halobacteroidaceae consist of species of the genus Acetohalobium, Haloanaerobacter, Halanaerobaculum, Halobacteroides, Halonatronum, Natroniella, Orenia, Selenihalobacter, and Sporohalobacter (Cayol et al., 1994b, 2000; Hedi et al., 2009; Liaw and Mah, 1992; Oren, 1983; Oren et al., 1984b, 1987; Simankova et al., 1993; Switzer Blum et al., 2001, 2009; Zeikus et al., 1983; Zhilina and Zavarzin, 1990; Zhilina et al., 1996, 2001). The most recent genus characterized among the halophilic fermentative bacteria is genus Halanaerobaculum, which was isolated from the hypersaline surface sediments of El-Djerid Chott (Tunisia) and characterized in 2009 (Hedi et al., 2009). The phylogenetic tree showing the phylogenetic position of type species of the halophilic fermentative bacterial genera based on 16S rRNA gene analysis is presented in Fig. 1. For comparison, the relationship of these bacteria to the newly identied non-fermentative members of the order Halanaerobiales, other members of the phylum Firmicutes and members of phyla Proteobacteria and Actinobacteria is shown. According to Oren (2006), the halophilic anaerobic bacteria are not closely related to the actinomycetes since they typically contain none of the characteristic nucleotides of the actinomycetes, and thus the tree was rooted using an actinomycete as an outgroup organism. Another typical genetic characteristic for halophilic fermentative bacteria is low content of guanosine and cytosine (G + C) of DNA. The G + C content of the type species is between 27 and 35 mol%, except for halothermophilic Ht. orenii and Nt. thermophilus with G + C contents of 40%.

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A.T. Kivist, M.T. Karp / Journal of Biotechnology 152 (2011) 114124

Fig. 1. A phylogenetic tree based on 16S rDNA sequence similarities between the type species of the halophilic fermentative genera (Table 1) and the newly identied halophilic non-fermentative genera of the order Halanaerobiales (Halarsenatibacter silvermanii and Selenihalobacter shriftii) to other members of the Firmicutes phylum (Bacillus subtilis, Clostridium butyricum, Lactobacillus plantarum, Ruminococcus avefaciens, and Thermobrachium celere), and to members of proteobacteria (Escherichia coli and Citrobacter freundii) and actinobacteria (Actinomyces bovis) phyla. The tree was constructed using multiple sequence alignment and Neighbour-Joining method (Larkin et al., 2007; Saitou and Nei, 1987) and rooted with A. bovis as an outgroup organism. Bootstrap values are shown at node points. Scale bar represents 10 nucleotide substitutions per 100 nucleotides.

A.T. Kivist, M.T. Karp / Journal of Biotechnology 152 (2011) 114124 Table 1 Taxonomic structure with order, family, genus, species, and subspecies of halophilic fermentative bacteria. Order Halanaerobiales Family Halanaerobiaceae Genus Halanaerobium Species H. acetethylicum H. alcaliphilum H. congolense H. fermentans H. kushneri H. lacurosei H. praevalensT H. saccharolyticum H. salsuginis Hc. cellulolsilyticaT Ht. oreniiT A. arabaticumT Han. chitinovoransT Han. lacunarum Han. salinarius Hab. tunisienseT Hb. elegans Hb. halobiusT Hn. saccharophilumT N. acetigenaT O. marismortuiT O. salinaria O. sivashensis Sp. lortetiiT T. berrensisT Nt. thermophilusT Subspecies

117

saccharolyticum senegalense

Halobacteroidaceae

Halocella Halothermothrix Acetohalobium Halanaerobacter

Halanaerobaculum Halobacteroides Halonatronum Natroniella Orenia

Clostridiales Natranaerobiales
T

Clostridiaceae Natranaerobiaceae

Sporohalobacter Thermohalobacter Natranaerobius

=type species of the genus.

3. Characteristics The requirements of halophilic fermentative bacteria for living are uncomplicated including high salt concentration (525%), obligate anaerobic conditions, and simple organic compounds (e.g. sugars or amino acids) available for carbon source and thus species have been isolated from a wide range of hypersaline environments including hypersaline lagoons and lakes, high temperature or alkaline hypersaline lakes, soda lakes, saltern ponds and solar salterns, hypersaline sulphur springs, oil brines and elds, and food products (Bhupathiraju et al., 1994, 1999; Cayol et al., 1994a,b, 1995, 2000; Hedi et al., 2009; Kobayashi et al., 2000; Liaw and Mah, 1992; Moun et al., 1999, 2000; Oren, 1983; Oren et al., 1984b, 1987; Ravot et al., 1997; Rengpipat et al., 1988; Simankova et al., 1993; Tsai et al., 1995; Zeikus et al., 1983; Zhilina and Zavarzin, 1990; Zhilina et al., 1991, 1996, 1997, 1999). The species of the order Halanaerobiales are obligate anaerobes but have not been reported being extremely oxygen-sensitive (Oren, 2006). Most of the halophilic fermentative bacteria are moderate halophiles growing optimally at salt contents between 50 and 150 g/l. However, few extremely halophilic representatives occur; Halanaerobium lacurosei, Hb. tunisiense, and A. arabaticum with optimal salt content for growth 200, 200220, and 150180 g/l, respectively. Many of the halophilic fermentative bacteria are relatively slow growing with doubling times from 2 to 8 h (Cayol et al., 1994b; Hedi et al., 2009; Liaw and Mah, 1992; Mesbah and Wiegel, 2008; Oren, 1983; Zeikus et al., 1983; Zhilina et al., 2001). However, few representatives are able to grow with doubling time under 1 h including T. berrensis (0.46 h), O. marismortui (0.67 h), and Hb. halobius (0.92 h) (Cayol et al., 2000; Oren et al., 1984b, 1987).

strain to strain and even from cell to cell among the same cultivation. The cells occur singly, in pairs or in groups of several cells. All the halophilic fermentative bacterial species are Gram-negative, with one exception. The newest member of the Halanaerobiales, Hb. tunisiense, stained Gram-positive even though otherwise its cell wall is Gram-negative-like with clear periplasm space and thin and wavy cell wall (Hedi et al., 2009). Most of halophilic fermentative bacteria are motile and agellated peritrichously. As an exception, H. praevalens is not motile and has not been reported containing agella and A. arabaticum is motile containing not peritrichous but subterminal agella (Zeikus et al., 1983; Zhilina and Zavarzin, 1990). The production of heat-resistant endospores of some members of Halanaerobiales has been reported. The spore-forming bacteria belong to the family Halobacteroidaceae and include genera Halonatrum, Natroniella, Orenia, and Sporohalobacter (Oren et al., 1987; Oren, 1983; Zhilina et al., 1996, 2001). In addition, rare sporeformation in A. arabaticum cells have been reported (Zhilina and Zavarzin, 1990).

3.2. Extremophiles among halophiles Most of the halophilic fermentative bacteria characterized thus far are mesophilic, with optimal temperature for growth between 35 and 45 C, and neutrophilic, with optimal pH for growth between 6 and 8. However, some members of the order Halanaerobiales grow in double or even triple extreme environments. Ht. orenii and T. berrensis are halothermophilic bacteria isolated from Tunisian salt lake and a solar saltern in France, respectively, having optimal temperature for growth 60 and 65 C, respectively. Molecular adaptations that are often connected with thermophilic proteins include a hydrophobic core that helps to exclude solvent from the inner regions of protein inhibiting unfolding, a small ratio of surface to volume that improves stability, decreased glycine content that limits the options for exibility and increases rigidity and together with high ionic interactions on the surface resists unfolding of protein by creating a rm network over the molecule

3.1. Physiology Characteristics of the type species of the halophilic fermentative genera are shown in Table 2. The halophilic fermentative bacteria share many common characteristics. For example, the cells are typically rod-shaped with the length of rods varying remarkably from

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Table 2a Characteristics of halophilic fermentative bacteria. Characteristic Cell morphology Cell size (m) Gram stain Motility Spores Temp. range ( C) Optimal temp. ( C) pH range Optimal pH NaCl range (g/l) Optimal NaCl (g/l) Doubling time (h) G + C (mol%) Substrates utilized Halanaerobium praevalens Rod 0.91.1 22.6 Negative 560 37 6.09.0 7.07.4 20300 125 4.0 27 Fructose, glucose, n-acetyl glucosamine, mannose, pectin Halocella cellulolsilytica Rod 0.40.6 3.812 Negative + 2050 39 5.58.5 7.0 50200 150 NRa 29 Cellulose, cellobiose, mannose, galactose, glucose, sucrose, sorbitol, starch Halothermothrix orenii Rod 0.40.6 1020 Negative + 4568 60 5.58.2 6.57.0 40200 100 5.3 40 Arabinose, cellobiose, fructose, galactose, glucose, melibiose, mannose, starch, ribose, xylose Thermohalobacter berrensis Rod 0.5 38 Negative + 4570 65 5.28.8 7.0 20150 50 0.46 33 Cellobiose, fructose, glucose, maltose, mannose, mannitol, sucrose, glycerol, N-acetylglucosamine, starch, pyruvate, bio-Trypticase Acetate, ethanol, H2 , CO2 Cayol et al. (2000) Natranaerobius thermophilus Rod 0.20.4 35 Positive 3556 53 8.310.6 9.5 90190 100135 3.5 40 Fructose, cellobiose, ribose, trehalose, trimethylamine, puryvate, Casamino acids, acetate, xylose, peptone Acetate, formatee Mesbah and Wiegel (2008) Acetohalobium arabaticum Rod 0.71 25 Negative + Rare NRa 47 3840 5.88.4 7.48.0 100200 150180 NRa 34 Betaine, formate, puryvate, lactate, casaminoacids, histidine, trimethylamine Halanaerobacter chitinovorans Rod 0.5 1.48 Negative + 2350 3045 NRa 7.0 29292 117176 2.47 35 Glucose, fructose, mannose, acetylglucosamine, sucrose, maltose, cellobiose, chitinb A.T. Kivist, M.T. Karp / Journal of Biotechnology 152 (2011) 114124

End-productsc Reference
a b c d e

Butyrate, acetate, propionate, H2 , CO2 Zeikus et al. (1983)

Acetate, ethanol, lactate, H2 , CO2 d Simankova et al. (1993)

Acetate, ethanol, H2 , CO2 Cayol et al. (1994b)

Acetate Zhilina and Zavarzin (1990)

Acetate, isobutyrate, H2 , CO2 Liaw and Mah (1992)

Not reported. The type strain utilizes chitin as a substrate. End-products in glucose unless otherwise mentioned. End-products in cellulose. End-products in sucrose.

A.T. Kivist, M.T. Karp / Journal of Biotechnology 152 (2011) 114124 Table 2b Characteristics of halophilic fermentative bacteria. Characteristic Cell morphology Cell size (m) Gram stain Motility Spores Temp. range ( C) Optimal temp. ( C) pH range Optimal pH NaCl range (g/l) Optimal NaCl (g/l) Doubling time (h) G + C (mol%) Substrates utilized Halanaerobaculum tunisiense Rod 0.71 413 Positivea 3050 42 5.98.4 7.27.4 140300 200220 2.1 34 Glucose, sucrose, galactose, mannose, maltose, cellobiose, pyruvate, starch Acetate, butyrate, lactate, H2 , CO2 Halobacteroides halobius Rod 0.5 1020 Negative + 3047 3745 NRb NRb 70187 88146 0.92 31 Glucose, fructose, sucrose, galactose, mannose, maltose, rafnose, puryvate, starch Ethanol, acetate, H2 , CO2 Halonatronum saccharophilum Rod 0.40.6 3.510 Negative + + 1860 3655 7.710.3 8.08.5 30170 70120 2.5 34 Glucose, fructose, sucrose, maltose, starch, glycogen, Nacetyl-d-glucosamine, peptone, yeast extract Acetate, formate, ethanol, H2 , CO2 Natroniella acetigena Rod 11.2 615 Negative + + 2842 37 8.110.7 9.710.0 100260 120150 NDb 32 Lactate, pyruvate, glutamate, ethanol, propanol Orenia marismortui Rod 0.6 313 Negative + + 2550 3645 NRb NRb 29176 29117 0.67 30 Glucose, fructose, mannose, sucrose, starch, glycogen

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Sporohalobacter lortetii Rod 0.52 2.515 Negative + + 2552 3745 NRb NRb 41146 8299 8 32 Glucose, fructose, maltose, sucrose, starch

End-productsc

Acetate

Acetate, ethanol, butyrate, formate, H2 , CO2 Oren et al. (1987)

Reference
a b c

Hedi et al. (2009)

Oren et al. (1984b)

Zhilina et al. (2001)

Zhilina et al. (1996)

Acetate, n-butyrate, isobutyrate, isovalerate, propionate, H2 Oren (1983)

Cell wall gram-negative-like. Not reported. End-products in glucose unless otherwise mentioned.

surface (Madigan and Oren, 1999). It has been suggested that high intracellular K+ concentrations, which are often found also in halophilic fermentative bacteria, along with DNA binding proteins and positive supercoiling increase the stability of DNA in high temperatures (Madigan and Oren, 1999). Yet another typical characteristic for thermophiles is the special phospholipid fatty acid prole of cell wall dominated by saturated fatty acids (Cayol et al., 1994b). Few strains have been characterized growing in hypersaline environments with high alkalinity: N. acetigena and H. alcaliphilum (Tsai et al., 1995; Zhilina et al., 1996). N. acetigena is an homoacetogenic bacterium that was isolated from the soda depositing Lake Magadi (Zhilina et al., 1996). The pH optimum of N. acetigena for growth is between 9.7 and 10.0. H. alcaliphilum, isolated from hypersaline Great Salt Lake, Utah, is an alkalitolerant bacterium since it achieves its optimal growth rate at neutral pH but is capable for growing up pH 10.0 (Tsai et al., 1995). It is of importance for alkaliphiles to maintain the cytoplasmic pH suitable for cellular functions. And thus, even though the external pH of the growth environment is high, the intracellular pH typically is near neutral (Mesbah and Wiegel, 2008). There are several strategies to remain the neutral intracellular pH in alkaliphilic conditions including increased production of acidic metabolites as fermentation products, increased activity in coupling H+ entry to ATP generation, increased amount and activity of monovalent cation/H+ antiporters, and other changes in properties of cell surface (Mesbah and Wiegel, 2008). Not only halophilic fermentative alkaliphilic bacteria but also halophilic fermentative alkalithermophilic bacteria have been characterized: Nt. thermophilus was isolated from soda lake Wadi An Natrum, Egypt and characterized in 2007 (Mesbah et al., 2007). The bacterium is capable for fermentative growth and has optimal pH for growth of 9.5, optimal temperature of 53 C, and optimal salt content between 100 and 135 g/l. When considering halophilic alkalithermophiles, all the three extremes discussed are combined and special adaptation of the cells is required.

3.3. Metabolism There are three kinds of fermentative metabolic routes for the members of the order Halanaerobiales based on the substrate utilized. The metabolic routes of halophilic anaerobic bacteria have been relatively unexplored thus far but the ones of halophilic archaea have been partly characterized in more detail (Falb et al., 2008). Most of the members are able to utilize a wide range of carbohydrates including a variety of sugars and glycerol. Typical fermentation products of carbohydrate metabolism are acetate, ethanol, hydrogen, and carbon dioxide. However, not all strains have been reported to produce ethanol (Cayol et al., 1994a; Ravot et al., 1997; Zhilina et al., 1992). In addition, fermentation products may include butyrate, lactate, propionate, formate, valerate, and 1,3-propanediol (from glycerol) (Hedi et al., 2009; Liaw and Mah, 1992; Oren, 1983; Oren et al., 1987; Simankova et al., 1993; Zeikus et al., 1983; Zhilina et al., 2001). Some species utilize amino acids as substrate of fermentation. For example, Sp. lortetii, a spore-forming bacterium, uses primarily amino acids as substrates instead of sugars which are utilized only poorly (Oren, 1983). Besides this, some strains are able to ferment mixtures of amino acids using the Stickland reaction, i.e. a reaction in which one amino acid is used as an electron source and another as electron acceptor resulting in production of organic acids, NH3 , and CO2 (Nisman, 1954). For example, Halanaerobacter salinarius is able to grow using the Stickland reaction with serine as an electron donor and glycine-betaine as an acceptor (Moun et al., 1999). The third fermentative route of metabolism includes production of acetate from organic substrates such as lactate, ethanol, pyruvate, glutamate, propionate, and glycine-betaine by homoacetogens. Homoacetogenic members of the Halanaerobiales are A. arabaticum and N. acetigena (Zhilina and Zavarzin, 1990; Zhilina et al., 1996). The main difference between the two homoacetogenic representatives is the ability of A. arabaticum for growing on hydrogen and carbon dioxide or carbon monoxide (Zhilina and Zavarzin, 1990).

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4. Survival methods To survive such extreme hypersaline conditions, halophiles need to have specic strategies for balancing osmotic pressure caused by extremely high ion concentration of the environment. Basically, there are two strategies to handle such high extracellular salt concentrations: salt-in and organic solutes-in strategies. In the salt-in strategy, cells maintain high intracellular salt concentrations and in organic solutes-in strategy cells accumulate organic compatible solutes, e.g. glycerol, glycine-betaine, trehalose, and sucrose (Roberts, 2005). Since all biological membranes are permeable for water, the main idea of both strategies is to maintain isoosmotic conditions within the cell. Roughly classied, saltin strategy is used by aerobic extremely halophilic archaea and halophilic fermentative bacteria, whereas organic solutes-in strategy is used by halophilic alga, halophilic methanogenic archaea, and halotolerant and halophilic aerobic bacteria. The Halanaerobiales, as halophilic fermentative bacteria, typically cope with the extreme living conditions by balancing the osmotic pressure caused by the high salt concentration of the environment with high intracellular ion concentrations. High intracellular ion content of Halanaerobium acetethylicum, H. praevalens, and Hb. halobius has been reported and no reports for organic compatible solutes being accumulated by halophilic fermentative bacteria exist (Oren, 2001; Oren et al., 1997; Rengpipat et al., 1988). Most often the salt accumulated in molar concentrations is consisted of K+ and Cl ions. However, in some cases, especially in the stationary phase of growth, the main inorganic cation of the cytoplasm has been observed to be Na+ (Oren et al., 1997; Rengpipat et al., 1988; Roberts, 2005). Although, Cl is by far the most relevant anion accumulated by halophiles using the salt-in strategy, also high concentrations of sulfate have been observed in some halophilic microorganisms (Ede et al., 2004). Energy in salt-in strategy energy is typically used for extruding Na+ and accumulation of Cl ions. Uptake of K+ is often considered as passive process but might also be an active energy requiring process in some microorganisms, e.g. in halophilic aerobic archaea Haloferax volcanii (Oren, 2001). K+ ions pass through cell membrane via uniport system as a response to membrane potential, whereas chloride ions are transferred via symport along with Na+ (Oren, 2001). Based on the knowledge of the transfer systems of halophiles, it was estimated that accumulation of 1.52 mol KCl would cost energy as much as 1 mol ATP (Oren, 1999, 2001). Although life at hypersaline environment is energetically expensive, the salt-in strategy used by halophilic fermentative bacteria is energetically remarkable cheaper than production and accumulation of organic compatible solutes in the organic solutesin strategy used by other halophilic bacteria. According to Oren (2001), the reason why halophilic fermentative bacteria prefer saltin strategy is their loss of affordability for the strategy of organic solutes. The intracellular systems of microorganisms using salt-in strategy are exposed for high salt concentrations and thus need to be well adapted for maintaining their functionality and stability. For example, the non-halophilic proteins tend to aggregate and become rigid at high salt, whereas the halophilic proteins are able to stay soluble and function properly (Mevarech et al., 2000). The proteomic studies on halophilic microorganisms have mainly been focused on halophilic archaea thus far, and only little is known about halophilic eubacterial representatives. The proteins of halophilic microbes maintaining high intracellular ion concentrations are typically highly acidic and thus negatively charged, and denature at salt concentrations under 1 M (Fukuchi et al., 2003; Mevarech et al., 2000). The negative charge of the halophilic proteins has essential role in maintaining the solubility and stabilizing the native structure (Mevarech et al., 2000). However,

Matheson et al. (1987) reported that especially high content of acidic amino acids in ribosomal A-protein of H. praevalens was not observed. Furthermore, the studies on halophilic archaeal malatedehydrogenase revealed the reason for requirement of high salt: The specic binding of salt ions to the protein stabilizes the native form but the binding afnity is low (Mevarech et al., 2000). Halothermophilic Ht. orenii is the rst and only member of halophilic fermentative bacteria completely sequenced thus far. The genetic information of thermohalophilic Ht. orenii revealed unexpectedly that the protein structure is similar to thermophilic proteins instead of halophilic proteins. This means that the proteins of Ht. orenii might not be salt-tolerant leading to suggestion that bacterium is the rst halophilic fermentative bacterium observed not using salt-in strategy but organic solutes-in strategy for survival in hypersaline environment (Mavromatis et al., 2009). Sucrose might act as the osmotic stabilizer of Ht. orenii since in genetic analyses the gene coding for sucrose phosphate synthase was found (Huynh et al., 2005). In addition, genes coding for transport system of glycine-betaine and l-proline across the cell membrane were found and thus, although not able to produce, might act as osmotic stabilizers obtained from environment (Mavromatis et al., 2009). However, in practice, no accumulation of compatible solutes or ionic compounds has been reported thus far. 5. Potential for biotechnology According to the best of our knowledge, no commercial application of halophilic fermentative bacteria is currently available and the studies are at experimental phase. The few studies on the potential of halophilic fermentative bacteria are listed in Table 3. 5.1. Halofermentation Halophilic fermentation is reasonable when sustainable substrates are being used and high-value products being produced. Renewable substrates, waste materials, and by-products are often considered as sustainable substrates. Most of the type species of the halophilic fermentative genera are able to utilize starch as a substrate. Starch is a renewable substrate as an energy-storing polysaccharide produced by green plants. Along with starch, also cellulose, a polysaccharide used as a main component of cell wall by green plants, is often considered as a sustainable substrate. Only one species, Halocella cellulolsilytica, among the halophilic fermentative bacteria identied thus far is able to degrade cellulose. However, several strains are capable for biodegradation of cellobiose, a derivative of cellulose. Glycerol is produced as a by-product in biodiesel industry and within the increase in biodiesel production an oversupply of glycerol has been created leading to the rapid decrease in price of glycerol (Ma and Hanna, 1999). In the future, glycerol might even be considered as a waste material, an ideal substrate for biotechnological applications. Technical glycerol often contains several heavy metals and salts which are growth inhibiting factors for the most of the microorganisms. Halophilic bacteria might possess potential for biotechnological applications utilizing technical glycerol due to the facts that high heavy metal resistances of several halophilic bacteria have been reported and salts cause no problem for halophiles (Nieto et al., 1989). The fermentation products of halophilic fermentative bacteria include hydrogen, carbon dioxide, acetic acid, ethanol, and 1,3propanediol. Hydrogen gas is considered as a potential energy carrier in the future. The other typical gaseous metabolite of the halophilic fermentative bacteria, CO2 , is used in food, oil, and chemical industry. The liquid products are considered industrially as follows: Acetic acid is an important substance mainly for chem-

A.T. Kivist, M.T. Karp / Journal of Biotechnology 152 (2011) 114124 Table 3 The potential biotechnological applications studied for halophilic fermentative bacteria. Bacterium Halanaerobium saccharolyticum subsp. saccharolyticum Halanaerobium lacurosei Halanaerobium praevalens Potential application Biological 1,3-PD production Product 1,3-PD Level 0.49 mol/mol glycerol Reference Kivist et al. (2010)

121

Sporohalobacter marismortui

Halanaerocium saccharolyticum subsp. senegalense Halanaerobium saccharolyticum subsp. saccharolyticum

a b c

Biological COD removal Biodegradation of nitro-substituted aromatic compounds Biodegradation of nitro-substituted aromatic compounds Biological hydrogen production

NRa Corresponding amines

94% of COD removedb 100% degradationc

Kapdan and Erten (2007) Oren et al. (1991)

Corresponding amines

100% degradationc

Oren et al. (1991)

H2

1.61 mol/mol glycerol

Kivist et al. (2010)

Biological hydrogen production

H2

0.62 mol/mol glycerol

Kivist et al. (2010)

Not reported. The initial concentration of COD 1900 mg/l. The initial concentrations of nitro-substituted aromatic compounds 50100 mg/l.

ical industry, ethanol is highly valued as a motor fuel or fuel additive and bioethanol, in particular, is one of the most commonly used renewable fuels nowadays, and 1,3-propanediol is a high-value-chemical for use of polymer industry. In our studies, 1,3-propanediol production using glycerol as a substrate was observed in Halanaerobium saccharolyticum subsp. saccharolyticum with the yield of 0.49 mol 1,3-PD/mol glycerol (Kivist et al., 2010). The halophilic 1,3-PD production was observed to be dependent on alkaline pH, as reported earlier for 1,3-PD production in general by Barbirato et al. (1997) and Forsberg (1987). In addition to the end-metabolites, the potential products of halophilic fermentation include salt-tolerant proteins and enzymes. 5.2. Food biotechnology Halophilic fermentation has been taken into advantage in production of salt-containing food. The fermentation products give taste, aroma, and avor. It has been reported that acetate as a fermentation product even protects the food from contamination with spoiling yeasts (Margesin and Schinner, 2001). Halophilic or halotolerant fermentative bacteria have been used in production of a wide variety of food products, such as fermented sh, shrimp, meat, fruits, and vegetables (pickles), Asian sh and meat sauces, rice noodles and ours, and Indonesian soy sauce (Margesin and Schinner, 2001; Rling and van Verseveld, 1996; Tanasupawat and Komagata, 1995). Most of the bacteria that have been reported being involved in food production are not obligate halophiles including species among genera Lactobacillus, Halobacterium, Halococcus, Bacillus, Pediococcus, and Tetragenococcus (Margesin and Schinner, 2001; Rling and van Verseveld, 1996; Tanasupawat and Komagata, 1995). In addition to the fermentation products, halophilic bacteria, though not fermentative, have been reported to provide for dietary supplements, such as polyunsaturated long-chain fatty acids, and colorants, such as -carotene (Asker and Ohta, 1999; Nichols et al., 1993; Nichols and Russell, 1999). Polyunsaturated fatty acids are vital for human beings. Traditionally, they have been added into food in form of sh oil, which, however, might give the food undesired taste or odor (Margesin and Schinner, 2001). 5.3. Biopolymers The production of biosurfactants and other biopolymers have been reported for non-fermentative halophilic bacteria, although not for fermentative bacteria (Yakimov et al., 1995). However, since

the halophilic fermentative strains remain relatively little studied, the case might be that the production occur but has not yet been studied. Biosurfactants are biopolymers that decrease surface tension increasing the motility of hydrophobic hydrocarbons. Thus, the biosurfactants have been studied for the use in bioremediation of oil-contaminated hypersaline soil or water (Banat et al., 2000). Another potential use of halophilic biosurfactants is in situ microbially enhanced oil recovery. Many of the petroleum reservoirs are hypersaline with high temperature, and thus especially the halothermophilic bacteria surviving in the conditions could be useful in the area (Margesin and Schinner, 2001). In addition to biosurfactants, other halophilic biopolymers with potential have been found: exopolysaccharides with high viscosity for in situ enhanced oil recovery, liposomes for medicines and cosmetics, and bioplastics (Margesin and Schinner, 2001). 5.4. Enzymes The enzymes of the halophilic fermentative bacteria are salttolerant and, actually, salt-requiring due to the high intracellular ion concentrations maintained for balancing the osmotic pressure in hypersaline environment. The active form of the proteins and enzymes are achieved at high salt, and the denaturation occurs at low salt. The requirement for salt might be a challenge for the production and handling of the proteins but also a possibility for using enzymes in processes containing high salt concentrations. The highest interest among the halophilic enzymes is on isomerases and hydrolases including amylases that catalyze the bioprosessing of starch and -galactosidases that catalyze the bioprosessing of lactose. Salt-requirering enzymes have been cloned and produced as inactive forms in Escherichia coli and thereafter successfully activated with increase of salt concentration (Cendrin et al., 1993). 5.5. Biological waste treatment and biodegradation The potential of halophilic anaerobic fermentative bacteria for use in anaerobic treatment of saline waste waters has been reported (Kapdan and Erten, 2007; Oren, 2006). Halophilic fermentative bacteria have many advantages over conventional biological treatment systems. They are usable in high salt concentrations, might have tolerance for several heavy metals, and degrade wide range of organic compounds (Bhupathiraju et al., 1994; Cayol et al., 1995; Nieto et al., 1989; Tsai et al., 1995; Zeikus et al., 1983). According to Oren et al. (1992), halophilic microorganisms are in major role in biodegradation of pollutants in hypersaline environments.

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The potential of two halophilic fermentative bacteria, H. praevalens and O. marismortui, for biodegradation of nitro-substituted aromatic compounds including nitrobenzene, o-nitrophenol, mnitrophenol, p-nitrophenol, nitroanilines, 2,4-dinitrophenol, and 2,4-dinitroaniline was observed by Oren et al. (1991) and the biodegradation of most of the nitro-substituted aromatic compounds (initial concentrations 50100 mg/l) was completed within 24 h. Other compounds that halophilic microorganisms have been reported to degrade include saturated and aromatic hydrocarbons by archaeal members, and aromatic compounds, organophosphorus compounds, and formaldehyde by eubacterial members (Oren et al., 1992). 5.6. Alternative energy We are now living a time, when environmental issues and especially climate change are common public concerns. The use of fossil fuels has several harmful effects on environment and on health, as well (Levin et al., 2004). In addition, fossil fuels are not renewable source of energy. These are reasons why there is an urgent and constant need for alternative energy sources. Bioethanol, biobutanol, and biohydrogen are renewable alternatives for future gasoline. By the time of writing, butanol production in halophilic fermentative bacteria has not been reported but many of the halophilic fermentative species have been reported to produce ethanol, however, according to the best of our knowledge, halophilic ethanol production has not been studied in more detail. Hydrogen is a renewable and clean source of energy and thus considered as a potential energy carrier of the future. Hydrogen that is produced on renewable routes, such as a product in biological metabolism, is furthermore considered as a viable energy carrier. Hydrogen has several advantages as an energy carrier: The heat value is high, hydrogen is easily combustible, and when combusted, water is the sole product. Hydrogen is thus considered as environmental friendly potential energy solution for the future. The biological hydrogen production in hypersaline environment would not only be sustainable but also carefully protected from contamination. It has been reported that halophilic hydrogen utilizing methanogens, which without high selection pressure tend to contaminate bioprocesses, are rare (Oren, 2001, 2002). Therefore the sterilization costs of the halophilic hydrogen producing bioprocess could be minimized also in high volumes. In our studies, hydrogen production using glycerol as a substrate of fermentation was optimized in batch experiments in terms of pH, substrate and salt concentration in the two subsp. of H. saccharolyticum (Kivist et al., 2010). The major effect of pH and glycerol concentration and the minor effect of the salt concentration on the end-metabolite production were observed (Kivist et al., 2010). The highest hydrogen yields were achieved with 2.5 g/l glycerol and 150 g/l salt at pH 7.4 (subsp. saccharolyticum, yield 0.6 mol/mol glycerol) and at pH 7.0 (subsp. senegalensis, yield 1.6 mol/mol glycerol) (Kivist et al., 2010). The hydrogen yield of subsp. senegalensis might have potential for practical applications after scale-up and bioprocess optimizations and metabolic engineering after genomewide sequencing could be applied to improve the yield of subsp. saccharolyticum. 6. Conclusions Halophilic fermentative bacteria form taxonomically and phylogenetically a coherent group excluding few of the halophilic double or triple extremophilic bacteria. Halophilic bacteria have specic strategies for balancing the osmotic pressure caused by high extracellular salt concentrations. The halophilic fermentative bacteria typically use strategy in which cells accumulate high intracellular

salt (mainly K+ and Cl ) concentrations. The halothermophilic fermentative bacteria might be an exception, since based on genetic information of halothermophilic Ht. orenii, it has been suggested that the proteome of the bacterium is not halotolerant and that the bacterium would use compatible solutes-in strategy in which compatible solutes are produced and/or accumulated by the cell for balancing the osmotic pressure. Using either of the described strategy, living in hypersaline environment is not energetically cheap. Halophilic fermentative bacteria, with few exceptions, are capable for using a wide variety of carbohydrates and sugars as substrates of metabolism. The most typical fermentation products are acetate, ethanol, H2 , and CO2 . High-value fermentation products include, in addition, 1,3-PD and the salt-tolerant proteins. Halophilic fermentative bacteria might have potential for biotechnology on several areas including food biotechnology, biological waste treatment, and alternative energy. Hypersaline environments provide a bioprocess for high selection pressure and thus the sterilization costs can be minimized. The scarce knowledge of these microorganisms calls for intense studies to understand the genetic regulation and thereby metabolic engineering of these fascinating microorganisms for new and improved biotechnological processes. Further studies are needed concerning the heavy metal resistances in halophilic fermentative bacteria, in terms of occurrence, genes involved, and gene regulation. The heavy metal resistances would enable the use of many unpuried waste materials and by-products, including technical glycerol, as a substrate. In addition, studies are needed on characterization, expression and regulation of the genes coding for hydrogenase and hydrolase enzymes. Salt-tolerant reporter proteins have already been found thereby allowing the studies of gene expression in halophilic organisms (Sheridan and Brenchley, 2000). It has been shown that conjugation based approaches provide possibilities for genetic engineering of halophilic microorganisms. Genetic engineering together with whole genome sequence data would enable targeted manipulation for improved production of valuable products of biotechnological interest. Acknowledgements The research was funded by the Tampere University of Technology Graduate School (A.T.K.), Suomen Luonnonvarain Tutkimussti (Foundation of Research of Natural Resources in Finland project number 1719/09) the Academy of Finland (GMHydro project no. 108623; rimikro project no. 126974) and The Nordic Energy Research (BIOHYDROGEN project no. 28-02). References
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