how could haloalkaliphilic microorganisms contribute to biotechnology?

1/39

How could haloalkaliphilic microorganisms contribute to biotechnology? 1

Baisuo Zhao1*

, Yanchun Yan1, Shulin Chen

2 2

1Graduate School, Chinese Academy of Agricultural Sciences, Beijing 100081, China 3

2Biological Systems Engineering, Washington State University, Pullman, WA 99164, USA 4

*Corresponding author: [email protected] 5

Tel: 86-10-82106784 6

Fax: 86-10-82106784 7

Running title: Haloalkaliphilic microorganisms contribute to biotechnology8

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

2/39

9

Abstract: 10

Haloalkaliphiles are microorganisms requiring Na+ concentrations of at least 0.5 M and alkaline pH 9 11

for optimal growth. Their unique features enable them make significant contributions to a wide array of 12

biotechnological applications. Organic compatible solutes produced by haloalkaliphiles such as ectoine and 13

glycine betaine are correlated to osmoadaptation and may serve as stabilizers of intracellular proteins, salt 14

antagonists, osmoprotectants, and dermatological moisturizers. Haloalkaliphiles are an important source of 15

secondary metabolites like rhodopsin, polyhydroxyalkanoates, and exopolysaccharides that play essential 16

roles in biogeocycling organic compounds. These microorganisms also can secrete unique exoenzymes 17

including proteases, amylases, and cellulases that are highly active and stable in extreme halo-alkaline 18

conditions and can be used for the production of laundry detergent. Furthermore, the unique metabolic 19

pathways of haloalkaliphiles can be applied in the biodegradation/biotransformation of a broad range of toxic 20

industrial pollutants and heavy metals, wastewater treatment, and biofuel industry. 21

Key words: 22

Haloalkaliphile, Compatible solutes, Secondary metabolites, Exoenzymes, 23

Biodegradation/biotransformation, Biofuel industry24

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

3/39

25

1. Introduction 26

Haloalkaliphiles are extremophilic microorganisms capable of robust growth in rigorous ecosystems 27

characterized by both high salinity and extreme alkalinity (Sorokin and Kuenen 2005a, 2005b). They are 28

found in all three domains of life: Archaea, Bacteria, and Eukarya. They are mostly confined to exceptionally 29

stable, naturally occurring hypersaline alkaline environments like soda lakes and soda deserts distributed in 30

various dry steppes and semidesert areas around the world. These include soda lakes of Siberian and Kulunda 31

Steppe in Russia, Mono Lake and Soap Lake in North America, Natron Lake and Magadi Lake in the East 32

African Rift Valley, Magadi Lake in Kenya, and the Wadi Natrun lakes in Egypt (Foti et al. 2007, 2008, 33

Horikoshi 1999, Sorokin and Kuenen 2005). Such extremely halo-alkaline habitats are caused by the 34

combined effects of topography, geochemistry, and climate (Grant and Mwatha 1989, Kulp et al. 2007, 35

Sorokin et al. 2008). Topography is responsible for hydrological phenomena leading to a permanently 36

enclosed body of water. Geochemistry determines the major ionic composition and total salt content of these 37

endorheic drainage basins. Climate may lead to the accumulation of salts when evaporation rates exceed 38

in-flow rates. Saline soda lakes are characterized by the presence of high levels of salts, where the cation is 39

Na+ and K

+ as well as anion is mainly Cl¯, CO3

2¯, HCO3¯, and SO4

2¯. A correspondingly low concentration of 40

both Mg2+

and Ca2+

cations results from carbonate precipitation. 41

Athalassohaline lakes containing high levels of sodium carbonate / sodium bicarbonate represent a 42

combined conditions of both a salinity exceeding that of sea water (~35 g/L) and up to saturation and a pH 43

value generally around 9 and potentially up to 11 (Shapovalova et al. 2008, Sorokin et al. 2011). Microbial 44

populations in such athalassohaline harsh habitats thrive to the extent that they can be observed without the 45

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

4/39

aid of a microscope as different shades of red, purple, and green, representing archaea, photosynthetic purple 46

bacteria, and cyanobacteria, respectively. Hyper-saline and alkaline environments can alternatively result 47

from human industrial processes, such as those involving mineral ore, petroleum refining, pulp and paper, 48

textile preparation, leather tanneries, food and potato processing units, calcium carbonate kilns, and detergent 49

manufacture which generate effluents containing NaOH, Ca(OH)2, etc. (Alnaizy 2008, De Graaff et al. 2011, 50

Jones et al. 1998). 51

In general, microorganisms growing optimally at a NaCl concentration at or above 0.2 M, which differ 52

from natronophiles inhabiting in sodium carbonate brines, can be referred to as halophiles (Oren 2006, 53

Ventosa et al., 1998). Depending on the previous authors’ definitions, microorganisms growing optimally 54

either at or above 9, 9.5, or 10 are termed alkaliphiles by most of researchers although this definition is 55

disputable (Horikoshi 2006; Krulwich 2006). So far, an absolute definition for what constitutes 56

haloalkaliphiles has been elusive in the literature. For this review, the authors wish to focus on 57

microorganisms requiring for optimal growth at least 0.5 M total Na+ concentrations in the form of NaCl or 58

pH-mediating sodium carbonate / bicarbonate, and alkaline pH values of at least 9 (Table 1). 59

The term ‘haloalkaliphilic’ was earlier used to describe a novel extremophilic archaeon, Natronomonas 60

pharaonis DSM 2160T (formerly known as Natronobacterium pharaonis) originally isolated from biotopes of 61

Abu Gabara Soda Lake of Wadi Natrun in Egypt (Soliman and Trüper 1982), which possesses both a salinity 62

of 36 % and an alkaline pH of 11 (Imhoff et al. 1978). Subsequently, two haloalkaliphilic archaeal isolates, N. 63

pharaonis SP1T (DSM 3395

T) and Natronococcus occultus SP4

T (DSM 3396

T), were revived from the soda 64

lake Magadi in Kenya (Kamekura et al. 1997, Tindall et al. 1984). Since then, microorganisms that habitat in 65

the combined conditions of both high salinity and extreme alkalinity have been a topic of fascination to 66

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

5/39

microbiologists. 67

The potential for the discovery and utilization of novel haloalkaliphiles and their products is immense, 68

as they provide unique physiological functions under extreme conditions that limit survivability of their 69

mesophilic counterparts. During the three last decades, there has also been an increase in interest and 70

investment to explore haloalkaliphiles as a precious source of organic compounds, stable exoenzymes, and 71

unique metabolic pathways with potential applications in various industrial processes. There are still many 72

interesting and unresolved questions with respect to how they may affect and even benefit human life. 73

Specifically, how could these haloalkaliphilic microorganisms contribute to biotechnology? The objective of 74

this review is therefore to summarize and focus on the recent developments pertaining to the potential for 75

haloalkaliphiles to ultimately contribute to the efforts of the biotechnology`industry in meeting expanding 76

market demand for haloalkaliphile-based applications. 77

2. Organic compatible solutes 78

The properties of haloalkaliphile-derived compatible solutes can be biotechnologically exploited for a 79

wide range of applications in cosmetology, medicine, and agriculture, etc. Ectoine and hydroxyectoine can 80

stabilize enzymes to prevent denaturation via desiccation and chemical agents, and to enhance protein folding 81

via chemical chaperones. They also can be used to enhance PCR performance (Schnoor et al. 2004) and DNA 82

microarray sensitivity and specificity (Mascellani et al. 2007). A low concentration of hydroxyectoine (10 to 83

25 mM) has been demonstrated to reduce DNA microarray background and improve hybridization efficiency 84

(Mascellani et al. 2007). Glycine betaine effectively protects against mutagenic compounds and 85

radiation-induced damage. It can protect plants against abiotic stress from salt, drought, heat, and chilling. 86

For example, glycine betaine can be exogenously applied to leaves and roots of agronomically imporant 87

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

6/39

crops, like rice and potato, to increase tolerance to various stresses (Sakamoto and Murata 2000). 88

Compared to chemical synthesis, the haloalkaliphilic biosynthesis and subsequent extraction and 89

purification of compatible solutes like ectoine and glycine betaine are relatively easier and higher yielding. 90

The intracellular concentration of ectoine reached 235 µg/mg dry cells of Methylophaga alcalica M39T 91

grown at 1.6 M Na+ and pH 9.5 (Doronina et al. 2003b). Recently, a moderate haloalkaliphile, Methylophaga 92

lonarensis MPLT, intracellularly accumulated ectoine (149–197 µg/mg dry cells) as primary osmoprotectant 93

when grown under 1–1.5 M NaCl and pH 9–10 (Antony et al. 2012). Ectoine production by batch cultures of 94

the haloalkaliphilic Thioalkalimicrobium aerophilum AL 3T was proportional to extracellular Na

+ 95

concentration in mineral media (Banciu et al. 2005). Here, ectoine accounted for 8.7 % of total dry weight at 96

1.2 M Na+ and pH 10. Furthermore, the haloalkaliphilic Methylophaga murata Kr3

T synthesized ectoine, 97

where intracellular ectoine concentration increased from 40 µg/mg dry cells at 0.57 M Na+ to 160 µg/mg dry 98

cells at 1.60 M Na+ (Doronina et al. 2005). Interestingly, the intracellular ectoine concentration at a low 99

temperature of 4 ºC was two-fold than at a mesothermal temperature of 29 ºC, which is beneficial for 100

industrial processes that recover more products at short low temperature stress. When grown at high salt 101

salinity, the Kr3T cells could withstand heating, repeated freeze-thaw cycles, and lyophilization without 102

adding any cryoprotectants, indicating that ectoine could protect whole cells intact and its protein and DNA 103

against denaturation. The haloalkaliphilic Methylophaga natronica Bur2T also produced ectoine under 104

saline-alkaline conditions (Doronina et al. 2003a). In a 1.5 L continuous laboratory fermenter, 105

Thioalkalivibrio versutus ALJ 15 accumulated 7.5 %, and 9 % of total dry weight of glycine betaine as the 106

major compatible solutes at 2, and 4 M Na+, respectively (Banciu et al. 2005). The haloalkaliphilic 107

Desulfonatronospira thiodismutans ASO3-1 accumulated 16 % of total dry cell mass of glycine betaine at 3 108

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

7/39

M total Na+ concentration (Sorokin et al. 2008a). Zhao and Wiegel investigated the accumulated compatible 109

solutes from the anaerobic halophilic alkalithermophilic bacterium Natranaerobius thermophilus 110

JW/NM-WN-LFT that grew optimally at 3.5 M Na

+, pH

55ºC 9.5, and 53 ºC. When grown at 52 °C and pH

55ºC 111

9.5, and when extra-cellular Na+ concentration was increased from 3.3 M to 4.5 M, the intracellular 112

concentration of glycine betaine in N. thermophilus increased more than three-fold from 0.437 M to 1.321 M. 113

However, the haloalkaliphiles are less efficient producers since they have to spend additional energy for pH 114

homeostasis when comparing with the neutraphilic halophiles for compatible synthesis production. Therefore, 115

publications involving hydroectoine, glutamate and other compatible solutes from haloalkaliphiles are limited 116

and to date have not yet practically investigated their biotechnological potential. 117

3. Secondary metabolites 118

Haloalkaliphilies can produce secondary metabolites of tremendous importance to biotechnology. These 119

include rhodopsin and the biopolymer of polyhydroxyalkanoate (PHA) and exopolysaccharides (EPS). 120

Haloalkaliphile-derived retinal proteins like halorhodopsins (hR) have many potential applications for the 121

manufacture of photochromic molecular materials used for data storage, light-induced color changing, 122

holographic films, instant photoelectrical response, nonlinear optical capability, etc. Bivin and Stoeckenius 123

(1986) reported that thirty eight haloalkaliphilic bacteria isolated from alkaline salt lakes in Kenya and the 124

Wadi Natrun in Egypt mainly possessed two photoactive retinal pigments. One pigment had spectroscopic 125

properties similar to those of hR. The other pigment had kinetics very similar to those of a sensory rhodopsin 126

(sR). Two types of rhodopsins, hR and phoborhodopsin (ppR; also known as sensory rhodopsin II), were 127

found in the haloalkaliphilic N. pharaonis SP1T (Duschl et al. 1990, Hirayama et al. 1992). The hR is a 128

light-driven chloride pump that maintains the effective level of chloride anions in the cell and has an 129

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

8/39

absorption maximum between 575 and 580 nm. The ppR has an absorption maximum occurring at 498 nm 130

and can be stably solubilized in several kinds of detergents (Hirayama et al. 1992).The mutant strain KM-1 131

generated from N. pharaonis DSM 2160T over produced ten-fold greater amounts of hR than that the 132

wild-type due to a point mutation from As-Asp324 to Asn in the bacteriorhodopsin activator homologues 133

(Ihara et al. 2008). 134

Polyhydroxyalkanoates (PHAs) that are accumulated intracellularly as energy storage compounds 135

comprise a large number of polyesters synthesized from renewable carbon sources like agriculture waste via 136

bacterial fermentation. Secreted haloalkaliphilic polymers can be considered as a raw material for 137

biodegradable plastics. Owing to their biocompatible and biodegradable characteristics, PHAs are materials 138

of great interest for pharmaceutical and clinical purposes, and are regarded as an alternative to 139

non-biodegradable plastics produced from fossil-based resources (Braunegg et al. 1998, Strazzullo et al. 140

2008). A PHA-producing haloalkaliphilic bacterium Halomonas profundus AT 1214T grew optimally at 3 % 141

(w/v) sea salts and at pH 8–9 (Simon-Colin et al. 2008). The PHA yield of strain AT 1214 T

was nearly 270 142

mg/L in sea salts broth. The chemical composition of the PHAs consisted of different proportions of 143

poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHV) and poly-3-hydroxybutyrate (PHB) depending on the 144

various carbon substrates used. Strain AT 1214 T

is thus a viable candidate for the cost-effective manufacture 145

of PHAs from by-products of many industries. A haloalkaliphilic bacterium Halomonas campaniensis 5AGT 146

also produced PHAs (Gambacorta et al. 2005). At 1 M NaCl and pH 9 in the presence of glucose as carbon 147

source, PHB production represented more than 10 % of the wet cell weight (Strazzullo et al. 2008). Several 148

haloalkaliphilic microbes belonging to the species of Halomonas campisalis could also accumulate PHAs 149

granules Strain MCM B-1027 accumulated 54 % of dry cell weight as PHB co-PHV when grown in a 150

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

9/39

production-medium containing 1 % (w/v) maltose at 0.51 M NaCl and pH 9 (Kulkarni et al. 2010). The PHA 151

accumulated by strain MCM B-1027 is a biodegradable co-polymer of both PHB (96 mol %) and PHV (4 152

mol %), has a melting temperature of 166.5°C and tensile strength of 18.8 MPa, which renders its potential 153

application as a packaging material (Kanekar et al. 2011). 154

Exopolysaccharides (EPSs) from haloalkaliphiles have unusual characteristics and functional activities 155

that can be exploited for applications in the food, pharmaceutical, and cosmetics industries. Knowledge of the 156

factors influencing haloalkaliphilic EPS production is unfortunately still very limited. The EPS produced by 157

haloalkaliphilic M. murata Kr3T was composed of carbohydrate and protein moieties that could stabilize the 158

cells and prevent them from drying (Doronina et al. 2005). Strain Kr3T represents a promising candidate for 159

cost-effective large-scale production of EPS as the result of the ability to use methanol, methylamine, 160

trimethylamine, and fructose as carbon and energy sources under a wide range of pH and salinity. 161

4. Exoenzymes 162

Momentum has recently gained in the study of the behavior of active haloalkaliphile-secreted 163

exoenzymes in the coupled stringent factors of high salt and alkaline pH conditions. Exoezymes derived from 164

haloalkaliphile are stable, functional, and efficient biocatalysts in organic solvents because the extracellular 165

surrounding medium environment that they are secreted into resembles non-aqueous systems due to the 166

reduction of water activity by high salt concentration. Therefore, haloalkaliphile-secreted exoezymes are 167

further being considered for manufacturing and bioremediation processes because they have catalytic ability 168

not only at high salinity and alkalinity, but also at the low-water activity of non-aqueous solvent or surfactant 169

environments. The sustainable production of fuels, chemicals, biopolymers, and materials from renewable 170

biomass resources like agricultural waste may also require haloalkaliphilic exoezymes that can withstand the 171

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

10/39

high salt and pH conditions of pretreatment and hydrolytic degradation steps. These may include proteases, 172

α-amylases, cellulases, and xylanases, etc (Table 2). With all things considered, compared to enzymes from 173

halophiles or alkaliphiles, literature pertaining to those from haloalkaliphiles so far is limited and the 174

following includes a review of such works. 175

4.1. Proteases 176

Proteases are enzymes that conduct proteolysis, beginning protein catabolism by hydrolyzing the peptide 177

bonds that link amino acids together in the polypeptide chain forming proteins. They are important 178

constituents of detergents and typically need to be active and stable in conditions of high salt and surfactant 179

concentrations, alkaline pH 9–11 that prevail under harsh washing conditions. Therefore, the 180

haloalkaliphile-derived proteases have recently received much attention for their superior stability under 181

otherwise destabilizing industrial conditions. 182

The potentially useful proteases from the haloalkaliphiles Bacillus species have been purified and 183

extensively characterized. Protease production and secretion from the haloalkaliphilic Bacillus sp. strain Vel 184

reached a maximum level of 410 U/ml at optimal growth conditions of 10 % (w/v) NaCl and pH 9 (Patel et al. 185

2006). The protease secretion occurred with an optimum at pH 8–9 and was 186 U/ml, 172 U/ml, and 158 186

U/ml at 1.71, 2.56, and 3.42 M NaCl, respectively. It was active at 0–0.17 M salt concentration, pH 8.5–12, 187

and was optimal at pH 10–11 (Gupta et al. 2005). The protease was stable in various surfactants (i.e. 0.1 % 188

SDS, 0.1 % Triton X-100, and 0.1 % Tween 80) and heavy metal ions (i.e. 5 mM Mn2+

, 5 mM Zn2+

5 mM 189

Mg2+

, and 5 mM Cu2+

). The stability of this protease at the poly-extremities of high pH, high salt and the 190

presence of detergent components and surfactants as well as metal ions makes this enzyme particularly 191

suitable for its application in detergent industries.192

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

11/39

193

The crude serine protease was secreted at 0.185 mg/ml by the haloalkaliphilic Bacillus sp.AH-6 and was 194

exceptionally resistant, retaining 65 %, 50 %, and 95 % of its original activity after 24 h incubation in 8 M 195

urea, 4 M guanidine-HCl, and 2 M guanidine-HCl, respectively (Dodia et al. 2008a). This protease exhibited 196

catalysis and stability at pH 8–13 and 0–4 M NaCl with an optimum coinciding with pH 9–11 and 0.15–0.2 197

M NaCl, and this was slightly enhanced by 0.2 % Tween-80 and 0.05 % Triton X-100 (Dodia et al. 2008b). 198

The NaCl salt enhanced the enzyme’s substrate affinity, as reflected by the increase in the catalytic constant 199

(Kcat). Salt requirement for optimal catalysis also increased with temperature. Interestingly, this protease 200

could regain stability and functionality in the presence of NaCl after chemically denaturing (Dodia et al. 201

2008a). Such rare phenomenon can be further investigated to enrich the body of knowledge regarding protein 202

stability and denaturation. Furthermore, this serine protease was highly stable in oxidizing-reducing agents 203

and commercial detergents. These attributes suggest that this enzyme can be used in detergent applications to 204

remove proteinaceous stains and deliver unique benefits otherwise unobtainable with conventional detergent 205

technologies (Dodia et al. 2008). 206

An extracellular protease from the haloalkaliphile Salinivibrio costicola 18AGT was also purified and 207

characterized (Lama et al. 2005, Romano et al. 2005). It showed optimal activity at 60 °C in the presence of 208

both 0.34 M NaCl and pH 8, with 80 % of residual activity at pH 9. The protease was slightly activated by 209

denaturing agents such as SDS (0.1 %) and urea (6.0 M). Karan et al. (2011) reported that 38 haloalkaliphilic 210

bacteria isolated from Sambhar Salt Lake (India) were able to secrete haloalkaliphilic proteases. Four of these 211

strains named EMB1, EMB2, EMB3, and EMB4 exhibited 28, 37, 21 and 11 U/ml protease activities in 212

gelatin broth, respectively. The crude protease from strain EMB2 belonging to the Geomicrobium genus 213

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

12/39

exhibited appreciable stability at NaCl concentrations up to 3.42 M (optimum at 0.85 M NaCl), temperature 214

up to 70 °C (optimum at 50 °C), pH 6–12 (optimum at pH 10), and up to 75 % (v/v) concentrations of various 215

organic solvents. For instance, the protease still retained 39 %, 91 %, 98 %, and 101 % of its original activity 216

in cyclohexane, ethanol, benzene, and n-dodecane after 72 h incubation, respectively. The protease also was 217

compatible with up to 2 % (w/v or v/v) of surfactants (SDS, CTAB, Tween 20, Tween 80, Triton X-100, and 218

Triton X-114) and up to 1 % (w/v) of commercial detergents, indicating its potential use for industrial 219

applications. 220

As proteases can not only catalyze hydrolysis but also synthesize peptide bonds, they may represent an 221

alternative to chemical synthesis. A chymotrypsin-like and solvent-tolerant serine protease (Nep) from the 222

haloalkaliphilic archaeon Natrialba magadii was purified and characterized (Giménez et al. 2000). The Nep 223

was active and stable at pH 8 in mixtures of 1.5 M Na+ and up to 30 % (v/v) aqueous-organic solvents that 224

included glycerol, dimethylsulfoxide (DMSO), N,N-dimethyl formamide, propyleneglycol, and dioxane 225

(Ruiz and De Castro 2007). Under the optimal combined conditions of 30 % DMSO, 1.5 M NaCl, and pH 8, 226

Nep catalyzed the synthesis of the tripeptide Ac-Phe-Gly-Phe-NH2 from Ac-Phe-OEt ester and Gly-Phe-NH2 227

amide substrates (Ruiz et al. 2010). The Nep gene from N. magadii was cloned and expressed heterologously 228

in Haloferax volcanii, which represented the first recombinant system for the secretion of a haloarchaeal 229

protease and resulted in high level production and activity of Nep protein that biocatalyzed peptide synthesis 230

under low-water activity conditions (De Castro et al. 2008). The high activity and stability of this 231

heterologously expressed protease make it a promising candidate for future applications (Ruiz et al. 2010). In 232

addition, a chymotrypsinogen β-like protease from N. pharaonis exhibited optimal activity at between 0.5 233

and 4 M NaCl, pH 10, and 61°C (Stan-Lotter et al. 1999). Another serine protease that had chymotrypsin-like 234

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

13/39

activity from N. occultus during the stationary phase was purified, and its activity and stability depended on 235

high salt concentrations of 1–2 M NaCl, a broad pH range of 5.5–12, and rather thermophilic conditions with 236

an optimal activity at 60 °C in the presence of 1–2 M NaCl (Studdert et al. 1997, Studdert et al. 2001). 237

4.2. α-amylases 238

α-amylases are enzymes that randomly hydrolyze starch molecules, and their demand is currently 239

increasing because of its great potential to be extensively applied in various biotechnological industrial 240

processes involving starch liquefaction for biofuels and the manufacture of detergent and pharmaceuticals. 241

However, the α-amylases typically isolated from non-haloalkaliphiles do not meet industrial requirements due 242

to their inability to withstand harsh inhibitory industrial conditions of high salt, surfactant, and detergent 243

concentrations. Therefore, isolation and exploration of novel haloalkaliphile-derived α-amylases with 244

desirable halo-, alkaline-, and thermo-stability is crucial to meet industrial demands. 245

The first reported haloalkaliphilic extracellular α-amylase was derived from Natronococcus sp. strain 246

Ah-36, an archaeon from Kenyan soda lake of Lake Magadii which could tolerate a wide salinity (1.4 M 247

NaCl–saturation), pH (8–10), and temperature (20–55°C) (Kobayashi et al. 1992). When strain Ah-36 was 248

aerobically cultivated at 3.5 M Na+ and pH 9, α-amylase activity of 0.01 U/ml was detected at 70 h in the 249

culture broth and reached a maximum of 0.12 U/ml after 90 h. This enzyme was active at 1–5 M NaCl and 250

was most stable at pH 6–8.6 with a maximum at pH 8.7 and 2.5 M NaCl. It cleaves α-1, 4 linkages 251

endolytically and hydrolyzes soluble starch, amylose, amylopectin, glycogen, and γ-cyclodextrin to produce 252

maltotriose of α-configuration as the major product. Therefore, biotechnological applications of α-amylase 253

include the treatment of saline water or waste solutions containing starch residues at halo-alkaline and 254

poly-stress conditions. Prakash et al. (2009) reported two kinds of the extracellular α-amylase, designated as 255

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

14/39

α-amylase I and α-amylase II, optimally produced by the strain Chromohalobacter sp. TVSP 10 when grown 256

on 0.5 % rice flour at 3.4 M Na+ and pH 9. These were halo-alkalistable and moderately thermophilic 257

α-amylases with maximal activity at 0–3.41 M NaCl, pH 9, and 65°C that efficiently hydrolyzed 258

carbohydrates to yield maltotetraose, maltotriose, maltose, and glucose as end products. Recently, a 259

haloalkaliphilic α-amylase that was stable in surfactants, oxidants, and detergents was extracellularly secreted 260

from the actinomycete Saccharopolyspora sp. A9 at 1.9 M salinity and pH 11 (Chakraborty et al. 2011). This 261

enzyme hydrolyzed starch primarily to glucose, maltose, and maltotriose. It retained 72 % and 33 % of its 262

original activity at 1.9 and 2.9 M NaCl after 48 h incubation, respectively. It also retained 22 % and 68 % of 263

its original activity at pH 7 and 12 after 48 h incubation, respectively, and 54 % of its original activity even at 264

100°C after 6 h incubation. Furthermore, the enzyme retained 90–95 % of its activity in 0.5 % (w/v) of the 265

surfactants Tween 40, Tween 60, Tween 80 and cholic acid for 6–48 h incubation, 42–84 % of its activity in 266

the presence of the commercial detergents (0.1 %, w/v) such as rin, surf, ariel, and tide after 7–90 days 267

incubation at 1.89 M Na+ and pH 11, and 75–100 % of its activity in 0.2–1.2 % (w/v) of the oxidizing agents 268

H2O2 and NaClO3. The haloalkaliphilic α-amylase’s amazing stability can be used in applications involving 269

starch liquefaction and the manufacture of detergents and pharmaceutical industries, where high and 270

inhibitory levels of salt, surfactants and detergents are encountered. 271

4.3. Cellulase and xylanase 272

Cellulase and xylanase are enzymes that catalyze the hydrolysis of cellulose and hemicellulose, 273

respectively. They are used as laundry detergent additives and to hydrolyze ligno-cellulosic material in the 274

paper mill industry. So far, a report on cellulase purified from the first anaerobic haloalkaliphilic cellulolytic 275

bacterium Clostridium alkalicellulosi DSM 17461T (formerly designated Clostridium alkalicellum) is the 276

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

15/39

only instance of haloalkaliphile-derived cellulase (Zvereva et al., 2006; Zhilina et al. 2005). C. alkalicellulosi 277

secreted cellulases with molecular masses of 75 and 84 kDa and high affinity for amorphous or crystalline 278

cellulose. Optimal activity of this cellulase for both microcrystalline and phosphoric acid-swollen cellulose 279

substrate occurred within the pH range 6–9, with more than 70 % of its original activity being retained at pH 280

9.2. The cellulase activity of strain Z-7026T was similar to those of individual clostridial cellulase, but 281

significantly lower than those of intact cellulosome of Clostridium thermocellum. Comparing to the majority 282

of fungal cellulases, it retained more than 50 % of cellulase activity towards microcrystalline cellulose MCC 283

at pH 5–10 and more than 75 % of activity towards phosphoric acid-swollen cellulose at pH 5–9.4. Therefore, 284

this alkaline cellulase offers various opportunities for practical applications (i.e. detergents) where possess the 285

high pH values. Since xylan is more soluble at alkali than neutral conditions, isolation of xylan-degrading 286

microbe and their enzymes from alkaline pH environments has become a greater priority. An alkalistable 287

xylanase was produced by the haloalkalophilic bacterium Staphylococcus sp. strain SG-13, isolated originally 288

from an alkaline soil sample, in wheat bran medium (Gupta et al. 2000). The purified xylanase activity of 289

strain SG-13 has dual pH optima of 7.5 and 9.2 and an optimum temperature of 50°C. This xylanase 290

exhibited a substrate binding capacity of 92 % for hardwood oatspelt xylan but no significant substrate 291

binding for softwood birchwood xylan, and such specificity could be attributed to structural differences of 292

xylan polymers. A promising and cost-effective process for the large-scale production of a halo-alkaline 293

xylanase by the haloalkalitolerant Bacillus pumilus GESF-1 using wheat straw as a carbon source was 294

demonstrated (Menon et al. 2010). Xylanase activity was increased up to 120 % in 1.28 M NaCl, respectively. 295

Approximately 87 and 73 % of original activity was retained in 1.71 and 2.56 M NaCl, respectively, and 296

30 % of original activity was retained at pH 10, respectively. In our laboratory, an anaerobic and xylanolytic 297

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

16/39

bacterium, designed Alkalitalea saponilacus SC/BZ-SP2T, was derived from Soap Lake, WA (Zhao and Chen, 298

2012). This organism grew at total Na+ concentration of 0.35–1.38 M (optimum at 0.69 M), pH 7.5–10.5 299

(optimum at pH 9.7), and temperature of 8–40 ºC (optimum at 35–37 ºC). It utilized xylan, but no cellulose, 300

from beech wood, birch wood, and oat spelt wood as the sole carbon and energy source under high salinity 301

and alkaline pH, respectively. This implies the secreted extracellular xylanase by strain SC/BZ-SP2 into the 302

growth medium which can be easily purified and used in the paper mill industry for xylan removal. 303

5. Biodegradation and biotransformation 304

Haloalkaliphiles have gained much consideration to treat biological waste treatment and toxic residues 305

from halo-alkaline industrial processes via biodegradation/biotransformation. These compounds include 306

hydrosulfide (HS¯) and sulfide (S2–

), nitriles, benzene, toluene, ethylbenzene and xylenes (BTEX), benzoate, 307

salicylate, and phenol. Among them, HS¯ and S2–

typically are the most dominant sulfur compounds of spent 308

caustics originating from petroleum refineries, which are characterized by sodium concentrations of 309

0.86–2.05 M and a pH value of 9 and greater (Alnaizy 2008, De Graaff et al. 2011). In such combined 310

extreme environments, the haloalkaliphilic sulfur-oxidizing bacteria (SOB) can completely oxidize complex 311

sulfide wastes to sulfate or partially oxidize them to elemental sulfur under low oxygen conditions. In 312

addition, haloalkaliphilic sulfate-reducing bacteria (SRB) can partially oxidize complex sulfide wastes to 313

elemental sulfur though reductive reactions. Partial sulfide oxidation to elemental sulfur under oxygen 314

limitation is more advantageous than complete oxidation to sulfate because it results in regeneration of 315

hydroxyl ions other than protons, thereby limiting the need for caustic absorbent, facilitating separation of 316

elemental sulfur from the final oxidation product, and enabling recirculation of the liquid phase (Sorokin et al. 317

2008b). Many of the sulfide-oxidizing bacteria (SOB) have not yet to be isolated and characterized, but those 318

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

17/39

belong to four genera of Gammaproteobacteria (i.e. Thioalkalimicrobium, Thioalkalispira, Thioalkalibacter, 319

and Thioalkalivibrio) (Sorokin et al. 2013). De Graaff et al. (2011) reported successful on-site biotreatment of 320

spent caustics from petroleum refineries as an alternative to the physico-chemical treatment using chemical 321

oxidation. For this, sulfide was completely biotransformed into sulfate by a haloalkaliphilic SOB belonging 322

to the genus Thioalkalivibrio at 0.8 M Na+ and pH 9.5 in a continuously fed system with sulfide loading rates 323

of 27 m mol L¯1 day¯

1 and a hydraulic retention time (HRT) of 3.5 days. Similarly, a haloalkaliphilic 324

sulfur-oxidizing mixed culture enriched from Xochimilco alkaline soils (Mexico) containing Thioalkalivibrio 325

and Halomonas strains was active from pH 8–11.5 with a maximum activity at pH 10.6 (Olguín-Lora et al. 326

2011). Clearly, these strains of the genus Thiolalkalivibrio offer great potential for efficient desulfurization in 327

industrial processes at extremely halo-alkaline conditions. A commercial Thiopaq biotechnological 328

application for oxidizing sulfide to elemental sulfur was successfully demonstrated in lab-scale bioreactors 329

inoculated by mix sediments from hypersaline soda lakes (Sorokin et al. 2008b), most of the extremely 330

haloalkaliphilic SOB belongs to the genus Thioalkalivibrio. Fifteen haloalkaliphilic sulfate-reducing bacteria 331

(SRB) were isolated from a soda lake and determined to belong to the genus Desulfonatronum and the genus 332

Desulfonatronovibrio (Sorokin et al. 2011). 333

Compounds containing a C ≡ N (nitrile) are important intermediates in various industrial processes and 334

as building blocks in stereo-selective organic synthesis. They are also natural products formed by cyanide by 335

cyanogenic plants or amino acids by anaerobes (Sorokin et al. 2007a, Sorokin et al. 2008c). Thiocyanate 336

(CNS¯) is produced as a natural product via biological cyanide detoxification processes and as a waste 337

product in coke and metal plants, at high salinity and alkalinity (Sorokin et al. 2001). Several haloalkaliphilic 338

bacteria, Halomonas nitrilicus, Marinospirillum sp., Bacillus alkalinitrilicus, Natronocella acetinitrilica, and 339

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

18/39

Nitriliruptor alkaliphilus, which used aceto-, propio-, butyro-, iso-butyro-, and valero-nitriles as carbon, 340

energy, and nitrogen sources via nitrile hydratase/amidase pathway, have been isolated (Sorokin et al. 2007a, 341

Sorokin et al. 2007b, Sorokin et al. 2008c). Additionally, several thiocyanate-oxidizing bacteria belonging to 342

SOB genus Thioalkalivibrio were retrieved using on thiocyanate (CNS¯) as electron donor (Sorokin et al. 343

2001, Sorokin et al. 2010). The haloalkaliphilic bacterium Thialkalivibrio thiocyanodenitrificans ARhD 1T 344

could anaerobically oxidized thiocyanate with nitrate as electric acceptor via the identified intermediate 345

cyanate (up to 0.45 mM) and N2O (0.1 mM) to finally form ammonia (65–75% of the metabolized SCN¯ 346

nitrogen) and sulfate (90–95% of the metabolized SCN¯ sulfur) in fed-batch culture at a total Na+ 347

concentration of 0.58 M and pH 9.6 (Sorokin et al. 2004). The halotolerant alkaliphilies Thioalkalivibrio 348

thiocyanoxidans ARh 2T were able to use and degrade thiocyanate as the sole energy and nitrogen source 349

with cyanate as a major intermediate. This organism could also oxidize sulfide, polysulfide, sulfur, and 350

tetrathionate (Sorokin et al. 2002). 351

A variety of toxic aromatic compounds, including BTEX (benzene, toluene, ethylbenzene, and xylenes), 352

benzoate, salicylate, and phenol, are present in petroleum and spent caustic, saline, and alkaline waste 353

streams from refineries (De Graaff et al. 2011). In addition, benzoate is present in food and dye-processing 354

effluents, and salicylate is a key intermediate formed during biodegradation of several polycyclic aromatic 355

hydrocarbons, such as naphthalene and phenanthrene. Combined, the aforementioned aromatic hydrocarbon 356

compounds account for approximately 65 % of bulk-scale chemical waste, are on the priority pollutants list 357

of Environmental Protection Agency (EPA), and can therefore be regarded as models for understanding their 358

biodegradation at the extreme salinity and alkalinity of industrial effluents. Li et al. (2006) first observed that 359

the biodegradations of benzene and its derivatives could simultaneously occur under dual extremes of high 360

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

19/39

salinity and alkalinity. An aerobic haloalkaliphilie Planococcus sp. strain ZD22, isolated from saline and 361

alkaline soils of the Daqing oil field in China, used benzene as a sole source of carbon and energy. It 362

completely degraded 2 mM benzene at NaCl concentrations of 0.86–3.42 M for 3 days with an optimum of 363

1.71 M, and at pH 7.5–11 with an optimum of pH 9.5. This organism degraded and used not only benzene, 364

toluene, ethylbenzene and o-xylene, but also chlorobenzene, bromobenzene, iodobenzene, and fluorobenzene. 365

Under halo-alkaline conditions (i.e. 0.8 M Na+ and pH 9.5), approximately 93 % of benzene in refinery spent 366

caustics could be removed by a mixed-culture microbial community belonging to the genera Marinobacter, 367

Halomonas and Idiomarina (De Graaff et al (2011). In aerobic batch experiments at salinity of 0.86, 1.50, and 368

1.71 M NaCl and pH 9, Halomonas campisalis 4AT used and degraded both benzoate and salicylate at 369

concentrations of 380 mg/L as carbon and energy sources via the ortho-degradation pathway, as 370

intermetabolites like catechol and cis, cis-muconate were detected (Oie et al. 2007). Three 371

2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterial isolates were revived from the highly saline and 372

alkaline Alkali Lake site in Oregon previously contaminated with 2,4-D production wastes (Maltseva et al. 373

1996). The most active of them, strain 1-18 belonging to the family Halomonadaceae , grew optimally on 374

2,4-D at Na+ concentrations of 0.6–1.0 M and pH 8.4–9.4 and degraded up to 3000 mg 2,4-D in 3 days. Strain 375

1-18 used the modified artho-cleavage pathway for 2,4-D degradation, as catechol 1,2-dioxygenase, 376

muconate cycloisomerase, and dienelactone hydrolase were detected. A haloalkaliphilic Nocardioides sp. 377

strain M6 was also isolated from a closed site near Alkali Lake in Oregon that was previously contaminated 378

by high levels of chloro-aromatic compounds (Maltseva and Oriel 1997). It was capable of using 379

2,4,6-trichlorophenol, 2,4-dichlorophenol and 2,4,5-trichlorophenol. These investigations will lead to 380

improved haloalkaliphile biotechnological applications for industrial wastewater treatment in high salt and 381

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

20/39

high pH systems. 382

Biological denitrification of nitrate-containing waste, such as that found in agricultural run-off and 383

industrial downstream purification steps, is an essential microbially-facilitated process involving a series of 384

intermediate oxide products (NO2–, NO

–, and N2O) of nitrate to ultimately generate gaseous nitrogen at high 385

halolakaline, anoxic, natural / artificial environments. For example, the solution resulting from the 386

replacement of nitrate ions with chloride after industrial ion exchange separation may contain nitrate 387

concentrations of up to 1 g/L at both salt concentration of 0.5–2.0 M and pH of 8–9 (Peyton et al. 2001). 388

Fourteen strains of haloalkaliphilic denitrifiers belonging to genus Halomonas were isolated from extremely 389

saline soda lakes and soils (Shapovalova et al. 2008). All isolates could anaerobically grow on nitrate and 390

nitrite, and in some cases, N2O in saturated sodium carbonate brines (4 M total Na+) and pH 10. When 391

Halomonas sp. AGD 3 anaerobically grew on nitrate at high salinity, high levels of nitrite accumulation 392

occurred. In contrast, no significant level of nitrite and N2O was observed at below 1 M total Na+, indicating 393

that nitrite reduction was a limiting step in the hypersaline denitrification process. A highly active and 394

cytochrome c nitrite reductase (ccNiR) was purified from the haloalkaliphilic Thioalkalivibrio nitratireducens 395

ALEN 2T (Sorokin et al. 2003) molecularly characterized, and could both reduce nitrite and hydroxylamine to 396

ammonia without production of any intermediates and catalyze reductive conversion of sulfite to sulfide 397

(Tikhonova et al. 2006). As a stable hexamer in solution with molecular mass of about 360 kDa, it exhibited a 398

significantly high maximal specific activity (4080 ± 240 µmol/min per mg of the protein) with a reaction rate 399

half-saturation constant Km corresponding to16.7±4.0 mM of nitrite. Recombinant expression and large-scale 400

production of this enzyme is a possibility for the biotechnology industry. Two obligately haloalkaliphilic 401

denitrifiers, Halomonas mongoliensis Z-7009T and Halomonas kenyensis AIR-2

T, were isolated from soda 402

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

21/39

lakes and capable of reducing nitrate nitrite or N2O as well as oxidizing sulfide (Boltyanskaya et al. 2007). 403

Using acetate, lactate, and glycerol electron donors, Halomonas campisalis 4AT completely reduced 0.5 mg/L 404

nitrate for 3 days at 2.14 M Na+ and pH 9 (Mormile et al. 1999; Peyton et al. 2001). 405

With increasing industrial activity, toxic heavy metal wastes have led to soil and groundwater 406

contamination throughout the globe. Additionally, the radioactive wastes from the experimental detonations 407

of nuclear weapons, the nuclear fuel cycle reprocessing in utility plants, and the medical use of isotopes 408

presents additional problems. The environmental toxicity of some heavy metals at neutral pH conditions can 409

be reduced via their insolubility at alkaline pH conditions. An anaerobic and extremely haloalkaliphilic 410

bacterium strain SLAS-1 was isolated from sediment where it grew via arsenate [As(V)] respiration, using 411

either lactate or sulfide as its electron donor (Oremland et al. 2005). It could reduce arsenate [As(V)] to 412

arsenite As(III) by oxidizing lactate to acetate and HCO3– at optimal salinity range of 20 g/l–saturation and 413

pH 9.5. Several pertechnetate [Tc(VII)O4–]-reducing haloalkaliphiliic Halomonas spp. isolated from 414

soda-lake environments can reduce Tc(VII)O4– to Tc(V), Tc(IV), and Tc(III) (Khijniak et al. 2003). Under 415

anaerobic conditions at 1.05 M Na+ and pH 10, an average of 62 % of the 0.25 mM pertechnetate was 416

reduced to Tc (IV) and Tc(V) by haloalkaliphiles after a 3 days incubation. The isolate SL1 belonging to 417

Halomonas genus from Soap Lake (WA) could reduce Cr(VI) to Cr(III) ion after a 25 days incubation, 418

resulting in the transformation of 75 % of the 0.1 mM Cr(VI) initially present. The maximum specific 419

reduction rate reached 1.6 ± 0.24×10-4

mM Cr(VI) day-1

mg-protein-1

(VanEngelen et al. 2008). 420

6. Biofuel industry 421

Much research on developing biofuels from renewable biomass has been motivated by the rising price of 422

petroleum, recognition of its ultimate depletion, and concerns over global climate change. Halopalkalihiles 423

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

22/39

have the potential to be applied in the production of biofuel products (e.g. hydrogen, ethanol and carboxylic 424

acids) and other biotechnological products using lignocellulosic biomass as feedstock. For instance, alkaline 425

pretreatment for lignin removal and partial neutralization may create a saline-alkaline environment that is 426

more easily tolerated by haloalkaliphiles during subsequent hydrolysis. In our laboratory, the fermentation 427

products from xylan of beech wood were quantified using 1 % (w/v) by SC/BZ-SP2T (Zhao and Chen, 2012). 428

The identified main end products in the stationary phase were propionate (29.5 mM), acetate (21.2 mM), and 429

minor amounts of burytate (1.51 mM), iso-valerate (0.41 mM) and ethanol (1.21 mM). The pulpmill 430

wastewater from wheat straw generated by alkaline extraction processes is comprised of large quantities of 431

lignin, mono- and di-aromatic compounds, and polysaccharides from cellulose and hemicellulose and very 432

high pH value. Four haloalkaliphilic bacteria of the genus Halomonas were isolated from pulpmill 433

wastewater with a high pH value of 11 (Yang et al. 2008). Among them, Halomonas sp. 19-A and Halomonas 434

sp. Y2 were able to use guaiacol, vanillin, dibenzo-p-dioxin, biphenyl and fluorene at pH 9.5 and 1.71 M 435

NaCl (Yang et al. 2008). These Halomonas species actively growing under extremely halo-alkaline 436

conditions make them promising agents for industrial processes. As a storage and transportation medium for 437

clean and renewable energy, hydrogen is playing an important role in a future energy economy. Elias (2011) 438

filed a patent for haloalkaliphile Halanaerobium hydrogenoformans (formerly designed H. sapolanicus) 439

isolated from Soap lake (WA). This bacterium produced hydrogen, acetate and formate as major metabolic 440

end-products using variety of C-5 and C-6 sugars derived from hemicellulose and cellulose, and grows 441

optimally at 1.2 M Na+ and pH 11 (Begemann et al. 2012; Brown et al. 2011). It can utilize swichgrass and 442

straw that pretreated at low temperature (RT and 55 ºC) than previous report to yield hydrogen with high 443

production rate (0.37 and 0.88 µmol H2/h/ml) in batch reactor. Therefore, it would be expected to offer large 444

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

23/39

hydrogen production from renewable biomass resources. 445

7. Conclusions and future perspectives 446

Haloalkaliphiles are an interesting and unique class of dual-extremophilic microorganisms thriving 447

under the combined harsh conditions of hyper salinity and extreme alkalinity. They represent the basis for 448

many new and potentially transformative biotechnological efforts aiming to provide novel enzymes and 449

compounds to meet rapidly growing industrial demands for numerous applications. The steady increase in the 450

isolation and identification of novel haloalkaliphiles by the scientific community is accelerating these efforts. 451

The most representative haloalkaliphilies were reviewed, focusing the discussion on their: commercial 452

source for compatible solutes, secondary metabolites, exoezymes, and potential for bioremediation and 453

biotransformation as well as biofuel. Although major advances have been made in the past three decades, 454

compared to the other extremophiles such as halophiles, alkaliphiles and thermophiles, our knowledge of 455

haloalkaliphiles and their exploitable physiology, metabolism, enzymology, and genetics is to date still 456

limited. This is partly due to the high salinity and alkalinity that require specialized media formulations and 457

non-corrodible equipment for large-scale haloalkaliphilic cultivation and subsequent downstream processing 458

for purification. Consequently, conventional industrial efforts for haloalkaliphiles to meet growing market 459

demand are currently hindered. In addition, molecular engineering breakthroughs to generate recombinant 460

mesophilic producers have not been very successful. 461

Further efforts are necessary to improve cultivation biomass concentrations, yields, and volumetric 462

productivities. These will greatly depend on the ability to redirect metabolic fluxes towards the production of 463

targeted products to optimize growth under potentially rigorous conditions. New breakthroughs will not only 464

aid in the discovery / understanding of haloalkaliphiles and enable their commercialization for a multitude of 465

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

24/39

applications but also allow for their use as sources of ideas for non-haloalkaliphiles. Recent biotechnological 466

advances involving high-throughput enzyme screening, protein engineering, genome sequencing, proteomics, 467

metabolomics, and cloning and heterologous expression in more easily cultivated mesophilic hosts will lead 468

to more novel haloalkaliphilic-based biotechnological applications with exciting new properties. 469

Acknowledgements 470

This paper was supported by the National Natural Science Foundation of China (no. 31370158) and the 471

Basic Research Fund of Chinese Academy of Agricultural Sciences (no. 0042014011). 472

References 473

Alnaizy, R. 2008 Economic analysis for wet oxidation processes for the treatment of mixed refinery spent 474

caustic. Environ Prog 27: 295-301. 475

Antony, C.P., Doronina, N.V., Boden, R., Trotsenko, Y.A., Shouche, Y.S., and Murrell, J.C. 2012 476

Methylophaga lonarensis sp. nov., a novel moderately haloalkaliphilic methylotroph isolated from the 477

soda lake sediments of a meteorite impact crater. Int J Syst Evol Microbiol 62:1613-1618. 478

Banciu, H., Sorokin, D.Y., Rijpstra, W.I, Sinninghe Damsté, J.S., Galinski, E.A., Takaichi, S., et al. 2005 479

Fatty acid, compatible solute and pigment composition of obligately chemolithoautotrophic alkaliphilic 480

sulfur-oxidizing bacteria from soda lakes. FEMS Microbiol Lett 243:181-187. 481

Begemann, M.B., Mormile, M.R., Sitton, O.C., Wall, J.D., and Elias, D.A. 2012 A streamlined strategy for 482

biohydrogen production with halanaerobium hydrogeniformans, an alkaliphilic bacterium. Front 483

Microbiol 3: 1-12. 484

Bivin, D.B., and Stoeckenius, W. 1986 Photoactive retinal pigments in haloalkaliphilic bacteria. J Gen 485

Microbiol 132:2167-2177. 486

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

25/39

Boltyanskaya, Y.V., Kevbrin, V.V., Lysenko, A.M., Kolganova, T.V., Tourova, T.P., Osipov, G.A., et al. 2007 487

Halomonas mongoliensis sp. nov. and Halomonas kenyensis sp. nov., new haloalkaliphilic denitrifiers 488

capable of N2O reduction, isolated from soda lakes. Microbiology (Russia) 76:739-747. 489

Brown, S.D., Begemann, M.B., Mormile, M.R., Wall, J.D., Han, C.S., Goodwin, L.A., et al. 2011. Complete 490

genome sequence of the haloalkaliphilic, hydrogen producing Halanaerobium hydrogenoformans. J 491

Bacteriol 193:3682-3683. 492

Chakraborty, S., Khopade, A., Biao, R., Jian, W., Liu, X.Y., Mahadik, K., et al. 2011 Characterization and 493

stability studies on surfactant, detergent and oxidant stable α-amylase from marine haloalkaliphilic 494

Saccharopolyspora sp. A9. J Mol Catal B-Enzym 68:52-58. 495

De Castro, R.E., Ruiz, D.M., Giménez, M.I., Silveyra, M.X., Paggi, R.A., and Maupin-Furlow, J.A. 2008 496

Gene cloning and heterologous synthesis of a haloalkaliphilic extracellular protease of Natrialba magadii 497

(Nep). Extremophiles 12:677-687. 498

De Graaff, M., Bijmans, M.F., Abbas, B., Euverink, G.J., Muyzer, G., and Janssen, A.J. 2011 Biological 499

treatment of refinery spent caustics under halo-alkaline conditions. Bioresour Technol 102:7257-7264. 500

Dodia, M.S., Bhimani, H.G., Rawal, C.M., Joshi, R.H., and Singh, S.P. 2008a Salt dependent resistance 501

against chemical denaturation of alkaline protease from a newly isolated haloalkaliphilic Bacillus sp. 502

Bioresour Technol 99:6223-6227. 503

Dodia, M.S., Rawal, C.M., Bhimani, H.G., Joshi, R.H., Khare, S.K., and Singh, S.P. 2008b Purification and 504

stability characteristics of an alkaline serine protease from a newly isolated haloalkaliphilic bacterium sp. 505

AH-6. J Ind Microbiol Biotechnol 35:121-131. 506

Doronina, N.V., Darmaeva, T.D., and Trotsenko, Y.A. 2003a Methylophaga natronica sp. nov., a new 507

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

26/39

alkaliphilic and moderately halophilic, restricted-facultatively methylotrophic bacterium from soda lake 508

of the Southern Transbaikal region. Syst Appl Microbiol 26:382-389. 509

Doronina, N.V., Darmaeva, T.D., and Trotsenko, Y.A. 2003b Methylophaga alcalica sp. nov., a novel 510

alkaliphilic and moderately halophilic, obligately methylotrophic bacterium from an East Mongolian 511

saline soda lake. Int J Syst Evol Microbiol 53:223-229. 512

Doronina, N.V., Li, T.D., Ivanova, E.G., and Trotsenko, I.A. 2005 Methylophaga murata sp. nov.:a 513

haloalkaliphilic aerobic methylotroph from deteriorating marble. Microbiology (Russia) 74:440-447. 514

Duschl, A., Lanyi, J.K., and Zimanyi, L. 1990 Properties and photochemistry of a halorhodopsin from the 515

haloalkalophile, Natronobacterium pharaonis. J Biol Chem 265:1261-1267. 516

Elias, D.A., Mormile, M.R., Begemann, M.B., and Wall, J.D. 2011 Fossil fuel-free process of lignocellulosic 517

pretreatment with biological hydrogen production. United States Patent Application 20110136196. 518

Foti, M.J, Sorokin, D.Y., Lomans, B., Mussman, M., Zacharova, E.E., Pimenov, N.V., et al. 2007 Diversity, 519

activity, and abundance of sulfate-reducing bacteria in saline and hypersaline soda lakes. Appl Environ 520

Microbiol 73: 2093-2100. 521

Foti, M.J., Sorokin, D.Y., Zacharova, E.E., Pimenov, N.V., Kuenen, J.G., and Muyzer, G. 2008 Bacterial 522

diversity and activity along a salinity gradient in soda lakes of the Kulunda Steppe (Altai, Russia). 523

Extremophiles 12:133-145. 524

Gambacorta, A., Romano, I., Giordano, A., Lama, L., and Nicolaus, B. 2005 Halomonas campaniensis sp 525

nov., a haloalkaliphilic bacterium isolated from a mineral pool of Campania Region, Italy. Syst Appl 526

Microbiol 28:610-618. 527

Giménez, M.I., Studdert, C.A., Sánchez, J.J., and De Castro, R.E. 2000 Extracellular protease of Natrialba 528

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

27/39

magadii:purification and biochemical characterization. Extremophiles 4:181-188. 529

Grant, W., and Mwatha, W. 1989 Bacteria from alkaline, saline environments. Recent advances in microbial 530

ecology Japan Scientific Societies Press, Tokyo, Japan:64-67. 531

Gupta, A., Roy, I., Patel, R.K., Singh, S.P., Khare, S.K., and Gupta, M.N. 2005 One-step purification and 532

characterization of an alkaline protease from haloalkaliphilic Bacillus sp. J Chromatogr A 1075:103-108. 533

Gupta, S., Bhushan, B., and Hoondal, G. 2000 Isolation, purification and characterization of xylanase from 534

Staphylococcus sp. SG-13 and its application in biobleaching of kraft pulp. J Appl Microbiol 88:325-334. 535

Hirayama, J., Imamoto, Y., Shichida, Y., Kamo, N., Tomioka, H., and Yoshizawa, T. 1992 Photocycle of 536

phoborhodopsin from haloalkaliphilic bacterium (Natronobacterium pharaonis) studied by 537

low-temperature spectrophotometry. Biochemistry 31:2093-2098. 538

Horikoshi, K. 1999 Alkaliphiles: some applications of their products for biotechnology. Microbiol Mol Biol 539

Rev 63:735-750. 540

Horikoshi, K. 2006 Alkaliphiles:genetic properties and applications of enzymes. Kodansha. Ltd, Tokyo. 541

Ihara, K., Narusawa, A., Maruyama, K., and Takeguchi, M. 2008 A halorhodopsin-overproducing mutant 542

isolated from an extremely haloalkaliphilic archaeon Natronomonas pharaonis. FEBS Lett 543

582:2931-2936. 544

Imhoff, J., Hashwa, F., and Trüper, H. 1978 Isolation of extremely halophilic phototrophic bacteria from the 545

alkaline Wadi Natrun, Egypt. Arch Hydrobiol 84:381-388. 546

Jones, B.E., Grant, W.D., Duckworth, A.W., and Owenson, G.G. 1998 Microbial diversity of soda lakes. 547


Kamekura, M., Dyall-Smith, M.L., Upasani, V., Ventosa, A., and Kates, M. 1997 Diversity of alkaliphilic 549

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

28/39

halobacteria:Proposals for transfer of Natronobacterium vacuolatum, Natronobacterium magadii, and 550

Natronobacterium pharaonis to halorubrum, Natrialba, and Natronomonas gen. nov., respectively, as 551

halorubrum vacuolatum comb. nov., Natrialba magadii comb. nov., and Natronomonas pharaonis comb. 552

nov., respectively. Int J Syst Bacteriol. 47:853-857. 553

Kanekar, P.P., Kulkarni, S.O., Jog, J.P., Patil, P.A., Nilegaonkar, S.S., Sarnaik, S.S., et al.. 2011 554

Characterisation of copolymer, poly (hydroxybutyrate-co-hydroxyvalerate) (PHB-co-PHV) produced by 555

Halomonas campisalis (MCM B-1027), its biodegradability and potential application. Bioresour Technol. 556

102:6625-6628. 557

Karan, R., Singh, S.P., Kapoor, S., and Khare, S.K. 2011 A novel organic solvent tolerant protease from a 558

newly isolated Geomicrobium sp. EMB2 (MTCC 10310):production optimization by response surface 559

methodology. N Biotechnol 28:136-145. 560

Khijniak, T.V., Medvedeva-Lyalikova, N.N., and Simonoff, M. 2003 Reduction of pertechnetate by 561

haloalkaliphilic strains of Halomonas. FEMS Microbiol Ecol 44:109-115. 562

Kobayashi, T., Kanai, H., Hayashi, T., Akiba, T., Akaboshi, R., and Horikoshi, K. 1992 Haloalkaliphilic 563

maltotriose-forming α-amylase from the archaebacterium Natronococcus sp. strain Ah-36. J Bacteriol 564

174:3439-3444. 565

Kulkarni, S.O., Kanekar, P.P., Nilegaonkar, S.S., Sarnaik, S.S., and Jog, J.P. 2010 Production and 566

characterization of a biodegradable poly (hydroxybutyrate-co-hydroxyvalerate) (PHB-co-PHV) 567

copolymer by moderately haloalkalitolerant Halomonas campisalis MCM B-1027 isolated from Lonar 568

Lake, India. Bioresour Technol. 101:9765-9771. 569

Kulp, T.R., Han, S., Saltikov, C.W., Lanoil, B.D., Zargar, K., and Oremland, R.S. 2007 Effects of imposed 570

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

29/39

salinity gradients on dissimilatory arsenate reduction, sulfate reduction, and other microbial processes in 571

sediments from two California soda lakes. Appl Environ Microbiol 73:5130-5137. 572

Krulwich, T.A. 2006 Alkaliphilic prokaryotes. In Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K-H., 573

Stackebrandt, E. (ed). The prokaryotes. Ecophysiology and Biochemistry. pp. 283-308. Springer, New 574

York. 575

Lama, L., Romano, I., Calandrelli, V., Nicolaus, B., and Gambacorta, A. 2005 Purification and 576

characterization of a protease produced by an aerobic haloalkaliphilic species belonging to the 577

Salinivibrio genus. Res Microbiol 156:478-484. 578

Li, H., Liu, Y.H., Luo, N., Zhang, X.Y., Luan, T.G., Hu, J.M., et al. 2006 Biodegradation of benzene and its 579

derivatives by a psychrotolerant and moderately haloalkaliphilic Planococcus sp. strain ZD22. Res 580

Microbiol 157:629-636. 581

Maltseva, O., McGowan, C., Fulthorpe, R., and Oriel, P. 1996 Degradation of 2,4-dichlorophenoxyacetic acid 582

by haloalkaliphilic bacteria. Microbiology 145:1115-1122. 583

Maltseva, O., and Oriel, P. 1997 Monitoring of an alkaline 2,4,6-trichlorophenol-degrading enrichment 584

culture by DNA fingerprinting methods and isolation of the responsible organism, haloalkaliphilic 585

Nocardioides sp. strain M6. Appl Environ Microbiol 63:4145-4149. 586

Menon, G., Mody, K., Keshri, J., and Jha, B. 2010 Isolation, purification, and characterization of haloalkaline 587

xylanase from a marine Bacillus pumilus strain, GESF-1. Biotechnol Bioprocess Eng 6:998-1005. 588

Mormile, M. R., Romine, M. F., Garcia, M. T., Ventosa, A., Bailey, T. J., Peyton, B. M. 1999 Halomonas 589

campisalis sp nov., a denitrifying, moderately haloalkaliphilic bacterium. Syst Appl Microbiol 22: 590

551-558. 591

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

30/39

Oie, C.S., Albaugh, C.E., and Peyton, B.M. 2007 Benzoate and salicylate degradation by Halomonas 592

campisalis, an alkaliphilic and moderately halophilic microorganism. Water Res 41:1235-1242. 593

Olguín-Lora, P., Le Borgne, S., Castorena-Cortés, G., Roldán-Carrillo, T., Zapata-Peñasco, I., Reyes-Avila, J., 594

et al. 2011 Evaluation of haloalkaliphilic sulfur-oxidizing microorganisms with potential application in 595

the effluent treatment of the petroleum industry. Biodegradation 22:83-93. 596

Oremland, R.S., Kulp, T.R., Switzer Blum, J., Hoeft, S.E., Baesman, S., Miller, L.G., and et al. 2005 A 597

microbial arsenic cycle in a salt-saturated, extreme environment. Science 308:1305-1308. 598

Oren, A. 2006 Life at high salt concentrations. In Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K-H., 599

Stackebrandt, E. (ed). The prokaryotes: Ecophysiology and Biochemistry. pp. 263–282. Springer, New 600

York. 601

Patel, R.K., Dodia, M.S., Joshi, R.H., and Singh, S.P. 2006 Purification and characterization of alkaline 602

protease from a newly isolated haloalkaliphilic Bacillus sp. Process Biochem 41:2002-2009. 603

Peyton, B.M., Mormile, M.R., and Petersen, J.N. 2001 Nitrate reduction with Halomonas campisalis:kinetics 604

of denitrification at pH 9.0 and 12.5 % NaCl. Water Res 35:4237-4242. 605

Prakash, B., Vidyasagar, M., Madhukumar, M., Muralikrishna, G., and Sreeramulu, K. 2009 Production, 606

purification, and characterization of two extremely halotolerant, thermostable, and alkali-stable 607

[alpha]-amylases from Chromohalobacter sp. TVSP 101. Process Biochem 44:210-215. 608

Romano, I., Gambacorta, A., Lama, L., Nicolaus, B., and Giordano, A. 2005 Salinivibrio costicola subsp. 609

alcaliphilus subsp. nov., a haloalkaliphilic aerobe from Campania Region (Italy). Syst Appl Microbiol 610

28:34-42. 611

Ruiz, D.M., and De Castro, R.E. 2007 Effect of organic solvents on the activity and stability of an 612

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

31/39

extracellular protease secreted by the haloalkaliphilic archaeon Natrialba magadii. J Ind Microbiol 613

Biotechnol 34:111-115. 614

Ruiz, D.M., Iannuci, N.B., Cascone, O., and De Castro, R.E. 2010 Peptide synthesis catalysed by a 615

haloalkaliphilic serine protease from the archaeon Natrialba magadii (Nep). Lett Appl Microbiol 616

51:691-696. 617

Sakamoto, A., and Murata, N. 2000 Genetic engineering of glycinebetaine synthesis in plants:current status 618

and implications for enhancement of stress tolerance. J Exp Bot 51:81-88. 619

Schnoor, M., Voss, P., Cullen, P., Böking, T., Galla, H.J., Galinski, E.A., et al. 2004 Characterization of the 620

synthetic compatible solute homoectoine as a potent PCR enhancer. Biochem Biophys Res Commun 621

322:867-872. 622

Shapovalova, A.A., Khijniak, T.V., Tourova, T.P., Muyzer, G., and Sorokin, D.Y. 2008 Heterotrophic 623

denitrification at extremely high salt and pH by haloalkaliphilic Gammaproteobacteria from hypersaline 624

soda lakes. Extremophiles 12:619-625. 625

Simon-Colin, C., Raguénès, G., Cozien, J., and Guezennec, J. 2008 Halomonas profundus sp. nov., a new 626

PHA producing bacterium isolated from a deep sea hydrothermal vent shrimp. J Appl Microbiol 627

104:1425-1432. 628

Soliman, G.S.H., and Trüper, H. 1982 Halobacterium pharaonis sp. nov., a new, extremely haloalkaliphilic 629

archaebacterium with low magnesium requirement. Zbl Bakt Hyg I Abt Orig C 3:318-329. 630

Sorokin, D.Y., Banciu, H., Robertson, L.A., Kuenen, J.G., Muntyan, M.S., and Muyzer, D.G. 2013 Halophilic 631

and haloalkaliphilic sulfur-oxidizing bacteria. In Rosenberg, DeLong, E., E.F., Lory, S., Stackebrandt, E., 632

Thompson, F. (ed). The Prokaryotes: Prokaryotic Physiology and Biochemistry. pp. 529-554. Springer 633

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

32/39

Berlin Heidelberg. 634

Sorokin, I.D., Kravchenko, I.K., Doroshenko, E.V., Boulygina, E.S., Zadorina, E.V., Tourova, T.P., et al. 2008 635

Haloalkaliphilic diazotrophs in soda solonchak soils. FEMS Microbiol. Ecol 65:425-433. 636

Sorokin, D.Y., and Kuenen, J.G. 2005 Haloalkaliphilic sulfur-oxidizing bacteria in soda lakes. FEMS 637

Microbiol Rev 29:685-702. 638

Sorokin, D.Y., Tourova, T.P., Lysenko, A.M., and Kuenen, J.G. 2001 Microbial thiocyanate utilization under 639

highly alkaline conditions. Appl Environ Microbiol 67:528-538. 640

Sorokin, D.Y., Kuenen, J.G., and Muyzer, G. 2011 The microbial sulfur cycle at extremely haloalkaline 641

conditions of soda lakes. Front Microbial 2: 44. 642

Sorokin, D.Y., Van Pelt, S., Tourova, T.P., and Muyzer, G. 2007a Microbial isobutyronitrile utilization under 643

haloalkaline conditions. Appl Environ Microbiol 73:5574-5579. 644

Sorokin, D.Y., Van Pelt, S., Tourova, T.P., Takaichi, S., and Muyzer, G. 2007b Acetonitrile degradation under 645

haloalkaline conditions by Natronocella acetinitrilica gen. nov., sp nov. Microbiology 153:1157-1164. 646

Sorokin, D.Y., Tourova, T.P., Lysenko, A.M., Mityushina, L.L., and Kuenen, J.G. 2002 Thioalkalivibrio 647

thiocyanoxidans sp. nov. and Thioalkalivibrio paradoxus sp. nov., novel alkaliphilic, obligately 648

autotrophic, sulfur-oxidizing bacteria capable of growth on thiocyanate, from soda lakes. Int J Syst Evol 649

Microbiol 52: 657-664. 650

Sorokin, D.Y., Tourova, T.P., Antipov, A.N., Muyzer, G., and Kuenen, J.G. 2004 Anaerobic growth of the 651

haloalkaliphilic denitrifying sulfur-oxidizing bacterium Thialkalivibrio thiocyanodenitrificans sp. nov. 652

with thiocyanate. Microbiology 150: 2435-2442. 653

Sorokin, D.Y., Tourova, T.P., Henstra, A.M., Stams, A.J.M., Galinski, E.A., and Muyzer, G. 2008a 654

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

33/39

Sulfidogenesis under extremely haloalkaline conditions by Desulfonatronospira thiodismutans gen. nov., 655

sp. nov., and Desulfonatronospira delicata sp. nov. a novel lineage of Deltaproteobacteria from 656

hypersaline soda lakes. Microbiology 154:1444-1453. 657

Sorokin, D.Y., Tourova, T.P., Sjollema, K.A., and Kuenen, J.G. 2003. Thialkalivibrio nitratireducens sp nov., 658

a nitrate-reducing member of an autotrophic denitrifying consortium from a soda lake. Int J Syst Evol 659

Microbiol 53: 1779-1783. 660

Sorokin, D.Y., Van den Bosch, P.L., Abbas, B., Janssen, A.J., and Muyzer, G. 2008b Microbiological analysis 661

of the population of extremely haloalkaliphilic sulfur-oxidizing bacteria dominating in lab-scale 662

sulfide-removing bioreactors. Appl Microbiol Biotechnol 80:965-975. 663

Sorokin, D.Y., Van Pelt, S., and Tourova, T.P. 2008c Utilization of aliphatic nitriles under haloalkaline 664

conditions by Bacillus alkalinitrilicus sp. nov. isolated from soda solonchak soil. FEMS Microbiol Lett 665

288:235-240. 666

Sorokin, D.Y., Kovaleva, O.L., Tourova, T.P., and Muyzer, G. 2010 Thiohalobacter thiocyanaticus gen. nov., 667

sp nov., a moderately halophilic, sulfur-oxidizing gammaproteobacterium from hypersaline lakes, that 668

utilizes thiocyanate. Int J Syst Evol Microbiol 60:444-450. 669

Sorokin, D.Y., Tourova, T.P., Kolganova, T.V., Detkova, E.N., Galinski, E.A., and Muyzer, G. 2011 Culturable 670

diversity of lithotrophic haloalkaliphilic sulfate-reducing bacteria in soda lakes and the description of 671

Desulfonatronum thioautotrophicum sp. nov., Desulfonatronum thiosulfatophilum sp. nov., 672

Desulfonatronovibrio thiodismutans sp. nov., and Desulfonatronovibrio magnus sp. nov. Extremophiles 673

15:391-401. 674

Stan-Lotter, H., Doppler, E., Jarosch, M., Radax, C., Gruber, C., and Inatomi, K.I. 1999 Isolation of a 675

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

34/39

chymotrypsinogen β-like enzyme from the archaeon Natronomonas pharaonis and other halobacteria. 676


Strazzullo, G., Gambacorta, A., Vella, F.M., Immirzi, B., Romano, I., Calandrelli, V., et al. 2008 678

Chemical-physical characterization of polyhydroxyalkanoates recovered by means of a simplified method 679

from cultures of Halomonas campaniensis. World J Microbiol Biotechnol 24:1513-1519. 680

Studdert, C.A., De Castro, R.E., Seitz, K.H., and Sánchez, J.J. 1997 Detection and preliminary 681

characterization of extracellular proteolytic activities of the haloalkaliphilic archaeon Natronococcus 682

occultus. Arch Microbiol 168:532-535. 683

Studdert, C.A., Herrera Seitz, M.K., Plasencia Gil, M.I., Sánchez, J.J., and De Castro, R.E. 2001 Purification 684

and biochemical characterization of the haloalkaliphilic archaeon Natronococcus occultus extracellular 685

serine protease. J Basic Microbiol 41:375-383. 686

Tikhonova, T.V., Slutsky, A., Antipov, A.N., Boyko, K.M., Polyakov, K.M., Sorokin, D.Y., et al. 2006 687

Molecular and catalytic properties of a novel cytochrome c nitrite reductase from nitrate-reducing 688

haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens. Biochim Biophys Acta. 689

1764:715-723. 690

Tindall, B., Ross, H., and Grant, W. 1984 Natronobacterium gen. nov. and Natronococcus gen. nov., two new 691

genera of haloalkaliphilic archaebacteria. Syst Appl Microbiol 5:41-57. 692

VanEngelen, M.R., Peyton, B.M., Mormile, M.R., and Pinkart, H.C. 2008 Fe (III), Cr (VI), and Fe (III) 693

mediated Cr (VI) reduction in alkaline media using a Halomonas isolate from Soap Lake, Washington. 694

Biodegradation 19:841-850. 695

Ventosa, A., Nieto, J.J., and Oren, A. 1998 Biology of moderately halophilic aerobic bacteria. Microbiol Mol 696

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

35/39

Biol Rev 62:504-544. 697

Yang, C., Cao, G., Li, Y., Zhang, X., Ren, H., Wang, X., Feng, J., et al. 2008 A constructed alkaline 698

consortium and its dynamics in treating alkaline black liquor with very high pollution load. PloS one 699

3:e3777. 700

Zvereva, E.A., Fedorova, T.V., Kevbrin, V.V., Zhilina, T.N., and Rabinovich, M.L. 2006 Cellulase activity of 701

a haloalkaliphilic anaerobic bacterium strain Z-7026, Extremophiles 10:53–60. 702

Zhao, B., and Chen, S. 2012 Alkalitalea saponilacus gen. nov., sp. nov., an obligately anaerobic, alkaliphilic, 703

xylanolytic bacterium from Soap Lake, Washington State, USA. Int J Syst Evol Microbiol 2012 704

62:2618-2623. 705

Zhilina, T., Kevbrin, V., Tourova, T., Lysenko, A., Kostrikina, N., and Zavarzin, G. 2005 Clostridium 706

alkalicellum sp. nov., an obligately alkaliphilic cellulolytic bacterium from a soda lake in the Baikal 707

region. Microbiology 74:557-566.708

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

36/39

Table 1. The potentially biotechnological applications of haloalkaliphilic microorganisms in this review. 709

Table 2. The exoenzymes from haloalkaliphiles in this review. 710

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

37/39

Table 1 711

Microorganism Na+ conc. (M)

growth range (opt.)

pH

growth range (opt.)

Temp. °C

growth range (opt.)

Potential application Reference

Methylophaga alcalica M39T nd– 1.71 (0.51–0.68) 7– 11 (9–9.5) 4– 35 (25–29) ectoine, glutamate Doronina et al. 2003b

Methylophaga lonarensis MPLT 0.01– 1.54 (0.09–0.34) 7– 10 (9–10) 20– 37 (28–30) ectoine, glutamate Antony et al., 2012

Methylophaga murata Kr3T 0.05– 3.0 (0.5–1.5) 6– 10 (8–9) 0– 42 (20–32) ectoine, glutamate Doronina et al. 2005

Methylophaga natronica Bur2T nd– 1.71 (0.34–0.51) 7– 11 (8.5–9) 4– 37 (25–29) ectoine, glutamate Doronina et al. 2003a

Thioalkalimicrobium aerophilum AL 3T 1.2– 1.5 (0.3–0.5) 7.5– 10.6 (9.5–10) nd– 41 (30) ectoine, glutamate Banciu et al., 2005, Robertson et al., 2001

Desulfonatronospira thiodismutans ASO3-1T 1.5– 4.0 (2.0–2.5) 8.5– 10.6 (9.5–10) nd– 43 (35) glycine betaine Sorokin et al., 2011

Natranaerobius thermophilus JW/NM-WN-LFT 3.1– 4.9 (3.3–3.9) 8.3– 10.6 (9.5) 35– 56 (53) glycine betaine Zhao et al., (Data not published)

Thioalkalivibrio versutus ALJ 15T nd– 4.3 (1.0–2.0) 7.5– 10.6 (10) nd– 47 (35) glycine betaine Banciu et al., 2005; Robertson et al., 2001

Natronomonas pharaonis SP1T 2.0– 5.1 (3.5) 8– 11 (8.5–9) nd– 53 (43–45) halorhodopsin (hR); phoborhodopsin (ppR) Duschl et al. 1990; Hirayama et al. 1992;

Natronomonas pharaonis DSM 2160T 2.0– 5.1 (3.42) 8– 11 (8.5–9) nd– 53 (45) halorhodopsin (hR) Ihara et al., 2008; Xu et al. 1999

Halomonas profundus AT1214T 0.34– 0.51 (nd) 8–9 (nd) 32– 37 (nd) poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHV);

poly-3-hydroxybutyrate (PHB)

Simon-Colin et al. 2008

Halomonas campaniensis 5AGT 0– nd (1.71) 7– 10 (9) 10– 43 (37) poly-3-hydroxybutyrate (PHB) Gambacorta et al. 2005; Strazzullo et al. 2008

Halomonas campaniensis MCM B-1027 0– 4.0 (1.0) 7– 11 (9) 4– 45 (30) hydroxybutyrate-co-hydroxyvalerate

(PHB-co-PHV) copolymer

Kanekar et al. 2011; Kulkarni et al. 2010

Methylophaga murata Kr3T 0.05– 3.0 (0.5–1.5) 6– 10 (8–9) 0– 42 (20–32) exopolysaccharide (EPS) Doronina et al. 2005

Bacillus sp. Vel 0–3.5 (1.7) 7.0–9 (8–8.5) 37 (nd) protease Patel et al. 2005; Patel et al. 2006

Bacillus sp.AH-6 nd 10 (nd) 37 (nd) protease Dodia et al. 2008a, 2008b

Salinivibrio costicola 18AGT 0.1–4.3 (2.0) 7–10.5 (9) 10–40 (30) protease Lama et al. 2005, Romano et al. 2005

Geomicrobium sp. EMB2 0.85–3.42 (2.05) 7–10 (8.5) 30 (nd) protease Karan et al

Natrialba magadii ATCC 43099T 2.0–5.1 (4.1) 8.5–11.5(9.5) 37 (nd) protease Tindall et al. 1980; Mwatha & Grant. 1993

Natrialba pharaonis DSM 2160T 4.0 (nd) 9.5 (nd) 37 (nd) protease Stan-Lotter et al. 1999

Natrialba occultus NCBM 2192T 3.59 (nd) 10 (nd) 37 (nd) protease Studdert et al. 1997, 2001

Natronococcus sp. Ah-36 1.37–5.13 (2.56–3.42) 8–10 (9) 20–55 (40–45) α-amylase Kobayashi et al. 1992

Chromohalobacter sp. TVSP 101 0.85–5.13 (3.42) 6–10 (9) 35–55 (37) α-amylase Prakash et al. 2009

Saccharopolyspora sp. A9 4 (nd) 11 (nd) 37–55 (55) α-amylase Chakraborty et al. 2011

Clostridium alkalicellulosi DSM 17461T 0.02–0.4 (0.15–0.3) 8–10.2 (9) 18–42 (35–40) cellulose, xylanase Zhilina et al. 2005; Zvereva et al. 2006

Staphylococcus sp. SG-13 0.43–2.56 (nd) 6–11 (8) 25–50 (37) xylanase Gupta et al. 2000

Bacillus pumilus GESF-1 0.85–2.56 (0.85) 8–10 (8) 37 (nd) xylanase Menon et al. 2010

nd, not determined in the publications.712

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

38/39

Continue Table 1 713

Microorganism Na+ concentration (M)

growth range (opt.)

pH

growth range (opt.)

Temp. °C

growth range (opt.)

Potential application Reference

Thioalkalivibrio spp. 0.8 (nd) 9.5 (nd) 35 (nd) sulfur-oxidizing De Graaff et al. 2011

Thioalkalivibrio spp. 0.8 (nd) 8– 11.5 (10.6) 35 (nd) sulfur-oxidizing Olguín-Lora et al. 2011

Halomonas spp. 0.6– 1.7 (nd) 7– 11 (9.8) 30 (nd) sulfur-oxidizing Olguín-Lora et al. 2011

Thioalkalivibrio spp. 2– 3 (nd) 10 (nd) 30 (nd) sulfid → elemental sulfur Sorokin et al. 2008b

Desulfonatronum spp. 0.2– 2.5 (0.5–1) 8.0– 10.4 (9.3–9.5) 30 (nd) sulfate-reducing Sorokin et al. 2011

Desulfonatronovibrio spp. 0.2– 1.75 (0.5) 8.5– 10.3 (9.5–10) 30 (nd) sulfate-reducing Sorokin et al. 2011

Thialkalivibrio thiocyanodenitrificans ARhD 1T 0.3– 1.8 (nd) 9.6– 10 (nd) 30 (nd) biodegradation of thiocyanate Sorokin et al. 2004

Thioalkalivibrio thiocyanoxidans ARh 2T 0.3– 4 .3 (0.5–1) 8.5– 10.5 (10.2) 30 (nd) biodegradation of thiocyanate; sulfur-oxidizing Sorokin et al. 2002

Planococcus sp. ZD22 0.08– 4.27 (1.71) 7.5– 11 (9.5) 2– 36 (20–25) biodegradation of benzene Li et al. 2006

Halomonadaceae sp. 1-18 0.6– 1 (nd) 6.5– 10 (9–9.5) biodegradation of 2,4-dichlorophenoxyacetic acid Maltseva et al. 1996

Nocardioides sp. M6 0– 1.35 (0.2–0.4) 8.8– 9.8 (9.2) 30 (nd) biodegradation of 2,4,6-trichlorophenol,

2,4-dichlorophenol, and 2,4,5-trichlorophenol

Maltseva and Oriel 1997

Halomonas spp. 4 (nd) 10 (nd) 30 (nd) denitrification Shapovalova et al. 2008

Thioalkalivibrio nitratireducens ALEN 2T 0.2– 1.5 (0.4–0.5) 8.5– 10 (9.5–10) 35 (nd) denitrification Sorokin et al. 2003

Halomonas mongoliensis Z-7009T 0.16– 2.2 (0.7–1.7) 8– 10.5 (8.5–9.6) 15– 50 (36–40) oxidization of sulfide and reduction of nitrous oxide Boltyanskaya et al. 2007

Halomonas kenyensis AIR-2T 0.04– 2.2 (0.5–1.2) 7.5– 10.6 (9.5) 15– 55 (36–40) oxidization of sulfide and reduction of nitrate Boltyanskaya et al. 2007

Halomonas campisalis 4AT 0.2– 4.5 (1.5) 6– 12 (9.5) 4– 50 (30) biodegradation of benzoate and salicylate,

reduction of nitrate

Mormile et al. 1999; Peyton et al. 2001;

Oie et al. 2007

Halanaerobacteriales sp. SLAS-1 3.42– 5.64 (4.27–5.64) 9– 10.5 (9.5) nd (30) arsenate [As(V)] → arsenite [As(III)] Oremland et al. 2005

Halomonas spp. 1.05 (nd) 10 (nd) 30 (nd) Tc(VII)O4– → Tc(V), Tc(IV) and Tc(III) Khijniak et al. 2003

Alkalitalea saponilacusSC/BZ-SP2T 0.35– 1.38 (0.44–0.69) 7.5– 10.5 (9.7) 8– 40 (35–37) production of propionate and acetate Zhao and Chen 2012

Halanaerobium hydrogenoformans ATCC PTA-10410T 0.81– 2.94 (1.58) 7.5– 12 (11) 33 (nd) production of biohydrogen Begemann et al. 2012; Brown et al. 2011

Elias et al. 2011

nd, not determined in the publications. 714

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

39/39

Table 2 715

Exoenzymes from haloalkaliphiles Na+ concentration (M)

range (opt.)

pH

range (opt.)

Temp.°C

range (opt.) Reference

Protease from Bacillus sp. Vel 0– 0.17 (0.03) 8.5– 12.0 (10–11) 5– 50 (37) Gupta et al. 2005

protease from Bacillus sp.AH-6 0– 4 (0.15–0.2) 8– 13 (9–11) 37– 80 (50) Dodia et al. 2008a, 2008b

Protease from Salinivibrio costicola 18AGT 0– 2.57 (0.34) 5– 12 (8) 5– 100 (50) Lama et al. 2005

Protease from Geomicrobium sp. EMB2 0– 3.42 (0.85) 6– 12 (10) 0– 70 (50) Karan et al. 2011

Protease from Natrialba magadii ATCC 43099T 0.5– 2 (1–1.5) 6– 12 (8–10) 0– 70 (60) Giménez et al. 2000

Protease from Natrialba pharaonis DSM 2160T 0– 4 (0.5–4) 6– 12 (10) 0– 70 (61) Stan-Lotter et al. 1999

Protease from Natrialba occultus NCBM 2192T

0.1– 4 (1–2) 5.5– 12 (7–9) 30– 70 (60) Studdert et al. 1997, 2001

α-amylase from Natronococcus sp. Ah-36 1– 5(2.5) 4.2– 10.5 (8.7) 30– 60 (55) Kobayashi et al. 1992

α-amylase from Chromohalobacter sp. TVSP 101 0– 5.12(0–3.41) 5– 10 (9) 30– 80 (65) Prakash et al. 2009

α-amylase from Saccharopolyspora sp. A9 0.60– 2.9(1.88) 4– 12 (10–12) 55– 100 (55–95) Chakraborty et al. 2011

Cellulase from Clostridium alkalicellulosi DSM 17461T nd 4– 11 (6–9) nd Zvereva et al. 2006

Pylanase from Staphylococcus sp. SG-13 nd 6.0– 10.5 (7.5–9.2) 10– 65 (50) Gupta et al. 2000

Xylanase from Bacillus pumilus GESF-1 0– 2.56 (0.85) 7– 13 (7–8) 30– 70 (40) Menon et al. 2010

nd, not determined in the publications 716

of 39C

an. J

. Mic

robi

ol. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

09/2

8/14

For

pers

onal

use

onl

y. T

his

Just

-IN

man

uscr

ipt i

s th

e ac

cept

ed m

anus

crip

t pri

or to

cop

y ed

iting

and

pag

e co

mpo

sitio

n. I

t may

dif

fer

from

the

fina

l off

icia

l ver

sion

of

reco

rd.

how could haloalkaliphilic microorganisms contribute to biotechnology?

Documents