Alkaliphiles Advanced article

Koki Horikoshi, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Article Contents
. Introduction
Kanagawa, Japan
. Distribution of Alkaliphiles

. Isolation
Alkaliphiles grow optimally or very well at pH values above 9, often between 10 and 12,
. Growth Conditions
but cannot grow, or grow only slowly, at more neutral pH values. The enzymology,
. Cell Walls
physiology, ecology, molecular biology and genetics of alkaliphiles, as well as industrial
. Flagella of Alkaliphiles
applications of these microorganisms have been investigated extensively and several
. Sodium Ion and Membrane Transport
enzymes have been put to use on an industrial scale.
. Mechanisms of Cytoplasmic pH Regulation

. Genetics
. Alkaline Enzymes

. Alkaline Cellulases

. Future of Alkaliphiles

Online posting date: 15th July 2008

Introduction even from acidic soil samples, and faeces (Horikoshi, 1991).
Haloalkaliphiles have been found in extremely alkaline sa-
The discovery of alkaliphiles was fairly recent. In 1968, line environments, such as the Rift Valley lakes of East
only 16 scientific articles on the topic could be found, since Africa and the western soda lakes of the USA (Jones et al.,
the first report of an alkaliphilic Bacillus strain by Vedder 1998). See also: Extremophiles; Halophiles
(1934). The use of alkaliphilic microorganisms has a long
history in Japan, since from ancient times indigo has been
naturally reduced under alkaline conditions in the presence Isolation
of sodium carbonate. Indigo from indigo leaves is reduced
by particular bacteria that grow under these highly alkaline Alkaliphiles
conditions in a traditional process called ‘indigo fermen-
tation’. The most important factor in this process is the Alkaliphilic microorganisms coexist with neutrophilic mi-
control of the pH value. Formerly, indigo reduction was croorganisms, as well as occupying specific extreme envi-
controlled only by the skill of the craftsman. Microbio- ronments in nature. Figure 2 illustrates the relationship
logical studies of the process, however, were not conducted
until the rediscovery of these alkaliphiles by the author in
1968 (Horikoshi, 1971a). Alkaliphiles remained little more
than interesting biological curiosities, and at this time no
further industrial application was attempted.

Distribution of Alkaliphiles
Growth

Alkaliphiles consist of two main physiological groups of

microorganisms: alkaliphiles and haloalkaliphiles. Alkali-
philes require an alkaline pH of 9 or more for their growth
and have an optimal growth pH of around 10 (Figure 1),
whereas haloalkaliphiles require both an alkaline pH (49)
and high salinity (up to 33% (w/v) NaCl). Alkaliphiles have
been isolated mainly from neutral environments, sometimes

ELS subject area: Microbiology

5 7 9 11
How to cite: pH
Horikoshi, Koki (July 2008) Alkaliphiles. In: Encyclopedia of Life Sciences
(ELS). John Wiley & Sons, Ltd: Chichester. Figure 1 The pH dependence of alkaliphilic microorganisms. Typical
DOI: 10.1002/9780470015902.a0000337.pub2 growth–pH dependence of neutrophilic and alkaliphilic bacteria are shown
with squares and solid circles, respectively.

ENCYCLOPEDIA OF LIFE SCIENCES & 2008, John Wiley & Sons, Ltd. www.els.net 1
 Alkaliphiles
106
Counts of alkaliphilic bacteria per gram soil
105
104
103
102
101
1 2
Figure 2 Distribution of alkaliphilic microorganisms in environments of
various pH.
Table 1 Media for aerobic heterotrophic alkaliphiles
Ingredients (g21)
Glucose
Soluble starch
Yeast extract
Polypeptone
K2HPO4
Mg2SO4
7H2O
Na2CO3
Agar
between the occurrence of alkaliphilic microorganisms
and the pH of the sample origin. An alkaline medium has to
be used to isolate alkaliphiles. Sodium carbonate is gener-
ally used to adjust the pH to around 10, because alkali-
philes usually require at least some sodium ions. Table 1
shows general-purpose alkaline media suitable for their
isolation. The frequency of alkaliphilic microorganisms
in neutral ‘ordinary’ soil samples has been shown to be
102 –105 per g of soil, which corresponds to 1/10–1/100 of
the population of the neutrophilic microorganisms. Recent
studies show that alkaliphilic bacteria have also been found
in deep-sea sediments collected from depths as great as the
10 898 m of the Mariana Trench. Furthermore, large num-
bers of alkaliphiles were detected in core samples obtained
from 350 m below sea floor near Japan islands. Many
different kinds of alkaliphilic microorganisms have been
isolated from a variety of environments, including bacteria
belonging to the genera Bacillus, Micrococcus, Pseudo-
monas and Streptomyces, and eukaryotes, such as yeasts
and filamentous fungi.
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The reasons why alkaliphilic microorganisms are found
in neutral, sometimes even acidic, environments could be
complex; there are two interesting and suggestive experi-
mental results. First, individual alkaliphilic microorgani-
sms are capable of altering the pH of their surrounding
environment by making it more alkaline. This has been
proved by cultivation of alkaliphiles in media at various
values of pH (7–8.5). Whatever the initial pH, the final pH
of these cultures became more alkaline, often reaching pH 9,
which is optimum for alkaliphile growth. Second, sequen-
tial transition of pH involving several different microor-
ganisms has been observed. One of the model experiments
showed a pH transition from 5 to 9, where a fungus,
Aspergillus oryzae (optimal pH for growth 4–5), a bacte-
rium, Bacillus circulans (optimal pH for growth 6–8) and
alkaliphilic Bacillus spp. were inoculated in a medium origi-
nally at pH 5. Initially, A. oryzae, which grows at acidic pH,
began to grow at pH 5. After cessation of growth, the pH of
3 4 5 6 7 8 9 10 11 12 the medium became more neutral owing to autolysis of the
pH of soil fungus. B. circulans, which then began to grow at neutral
pH, produced a fungal cell wall-lytic enzyme and increased
the pH to 8, which allowed the growth of the alkaliphilic
Bacillus strain. This experiment thus demonstrated the
potential for growth of alkaliphiles in a medium with an
initial pH of 5. See also: pH and Buffers; Fungal Physiology
Horikoshi-I Horikoshi-II
10 – Haloalkaliphiles
– 10 The most remarkable examples of naturally occurring
5 5 alkaline environments are soda deserts and soda lakes.
5 5 Extremely alkaline lakes, for example, Lake Magadi in
1 1 Kenya and the Wadi Natrun in Egypt, are probably the
0.2 0.2 most stable highly alkaline environments on Earth, at a
10 10 consistent pH of 10.5–12.0 depending on site. In these
20 20 lakes, Mg2+ and Ca2+ precipitate as carbonate salts; thus
very low levels of these ions are present in a soluble form, an
unusual situation for the aquatic environment. In these
environments, sodium carbonate is the major source of
alkalinity; accordingly, this chemical is again usually used
in the isolation of microorganisms that are capable of
growth under these alkaline conditions. Many organisms
isolated from alkaline and highly saline environments such
as soda lakes also require high salinity, which is achieved by
adding NaCl to the isolation medium. In particular, hyper-
saline soda lakes are populated by alkaliphilic represent-
atives of halophilic archaea. These red-pigmented bacteria
and the archaeans reach numbers of 107 –108 per mL in
soda lake brines. Other haloalkaliphiles include Bacillus
spp. and sometimes extraordinary blooms of halophilic
photosynthetic bacteria of the genera Ectothiorhodospira
and Halorhodospira have been reported in Lake Magadi
and the Wadi Natrun. Recently, by using a metagenomic
approach, several enzyme genes were isolated directly from
the Kenyan soda lakes, Lake Elmenteita and Crater Lake.
Novel genes in high alkaline-salts environmental DNA
(deoxyribonucleic acid) libraries will be good candidates
for future industrial applications.
 Alkaliphiles

Growth Conditions Table 2 Intracellular pH values in alkaliphilic Bacillus strains

at different external pH values
Alkaliphiles require alkaline conditions and sodium
ions for growth and alkaliphilic Bacillus spp. also require External Internal
such for sporulation and endospore germination. Sodium Microorganisms pH pH Method
ion-dependent uptake of nutrients has also been re- Bacillus alcalophilus 8.0 8.0 Methylamine
ported in alkaliphiles. Many alkaliphiles require complex 9.0 7.6
nutrients for growth; a few alkaliphilic Bacillus strains can 10.0 8.6
grow in simple minimal media containing, for example, 11.0 9.2
glycerol, glutamic acid or citric acid. Cultivation temper- B. firmus RAB 7.0 7.7 DMO
atures are usually in the range 20–558C for most alkali- 9.0 8.0
philes. Haloalkaliphiles isolated from alkaline hypersaline 10.5 9.4 Methylamine
lakes require alkaline media containing up to 20% (w/v) Bacillus strain 7.5 8.5 DMO
NaCl. Thermophilic anaerobic spore-forming alkaliphiles, YN-2000
such as alkaliphilic Clostridium strains, have been isolated 8.5 7.9
from sewage plants. See also: Bacterial Reproduction and 9.5 8.1
Growth 10.2 8.4 Methylamine
B. halodurans 7.0 7.3
C-125 intact cells
Internal pH 7.5 7.4
8.0 7.6
Most alkaliphiles have their optimal growth pH at around 8.5 7.8 BCECF
10, which is the most significant difference from well-in- 9.0 7.9
vestigated neutrophilic microorganisms. Differences be- 9.5 8.1
tween internal pH and environmental pH affect the 10.0 8.2
transportation of ions and substances through the plasma 10.5 8.4
membrane. Internal cytoplasmic pH can be estimated from B. halodurans 7.0 7.5
the optimal pH of intracellular enzymes. For example, a- C-125 protoplasts
galactosidase from an alkaliphile, Micrococcus sp. strain 8.0 7.9
31–2, had its optimal catalytic pH at 7.5, suggesting that 8.5 8.2
the internal pH is around neutral. Furthermore, the cell- 9.0 8.4
free protein synthesis systems from alkaliphiles optimally 9.3 8.6
incorporate amino acids into protein at pH 8.2–8.5, only
0.5 pH units higher than that of neutrophilic Bacillus Note: DMO, 5,5-dimethyl-2,4-oxazolidinedione.
subtilis.
Another method of measuring internal pH is to measure
the distribution inside and outside cells of compounds that the cell wall may play a key role in protecting the cell from
have different forms at different pH values, provided they alkaline environments. Components of the cell walls of sev-
are not actively transported by cells. A weakly alkaline eral alkaliphilic Bacillus spp. have been investigated in com-
compound, methylamine or bis(carboxyethyl)carboxy-flu- parison with those of the neutrophilic B. subtilis. In addition
orescein (BCECF), can be used for this purpose at alkaline to peptidoglycan, alkaliphilic Bacillus spp. contain certain
pH. Calculation of the distribution of these compounds acidic polymers. Hydrolytic analysis of the acidic com-
showed that the alkaliphiles had a lower internal pH than pounds yielded galacturonic acid, glucuronic acid, glutamic
that of the external environment. The internal pH was acid, aspartic acid and phosphoric acid. There was a clear
maintained at around 8, despite high external pH (8–11) as relationship between cell wall composition and pH-growth
shown in Table 2. Therefore, one of the key features in al- optimum characteristics, and alkaliphilic Bacillus strains
kaliphily is associated with the cell envelope, which dis- could be divided into three groups on the basis of acidic
criminates and maintains the intracellular neutral components (Aono and Horikoshi, 1983) as described in
environment separate from the extracellular alkaline envi- Table 3. Alkaliphilic Bacillus strains in group 1 (Bacillus
ronment. See also: Intracellular pH Measurement pseudofirmus 2b-2 etc.) contain teichuronic acid in the cell
walls and cannot grow at neutral pH, whereas the cell walls
of group 2 strains contain large amounts of acidic amino
Cell Walls acids and uronic acids. In contrast, the walls of group 3
organisms contain teichoic acid. Even though most of the
strains in group 2 (Bacillus halodurans C-125 etc.) can grow
Acidic polymers in the cell walls
at neutral pH, the amount of acidic amino acids and uronic
Since the protoplasts of alkaliphilic Bacillus strains lose their acids found in cell walls from bacteria grown at neutral pH
stability in alkaline environments, it has been suggested that was much smaller than in those grown at an alkaline pH.

ENCYCLOPEDIA OF LIFE SCIENCES & 2008, John Wiley & Sons, Ltd. www.els.net 3
 Alkaliphiles
Table 3 Cell wall composition of alkaliphilic Bacillus strains
Group Components of cell walls
1 High in glucuronic acid, teichuronic acid and
hexosamine
2 High in glutamic acid, aspartic acid, galacturonic
acid, glucuronic acid and teichuronic acid
3 Presence of phosphoric acid, similar to B. subtilis
Therefore, it is likely that the acidic components in the out-
ermost layer of the group 2 bacteria have a function in sup-
porting growth at alkaline pH. The negative charges on the
acidic nonpeptidoglycan components may give the cell sur-
face its ability to adsorb sodium and hydronium ions and
repulse hydroxide ions, and as a consequence enable the cells
to grow in alkaline environments. See also: Bacterial Cell
Wall; Gram-type Positive Bacteria
Peptidoglycan composition
The peptidoglycans of alkaliphilic Bacillus spp. appeared
to be similar to that of B. subtilis. However, their compo-
sition was characterized by an excess of hexosamines and
amino acids in the cell walls as compared to neutrophilic
B. subtilis. Glucosamine, muramic acid, D and L-alanine,
D-glutamic acid, meso-diaminopimelic acid and acetic acid
were found in hydrolysates. Although some variation in the
amide content among the peptidoglycans from alkaliphilic
Bacillus strains was found, the variation in pattern was
similar to that known in neutrophilic Bacillus spp. See also:
Peptidoglycan
Flagella of Alkaliphiles
Some alkaliphiles are motile by flagella. This flagella-
induced motility is driven by a sodium-motive force instead
of the proton-motive force typical of neutrophiles (Hirota
et al., 1981). Alkaliphiles require Na+ for motility and are
most motile at pH 9.0–10.5, with no motility observed at
pH 8, nonmotile cells grown initially at pH 7 possessing a
few straight flagella. See also: Bacterial Flagella
The flagellin protein gene of B. halodurans C-125 was
cloned and expressed in the Escherichia coli system.
Sequencing this gene revealed that it encodes a protein of
272 amino acids. This flagellin shares high homology with
other known flagellins, such as B. subtilis 168 etc. Then,
Ito et al. (2004) identified the stator-force generator that
drives Na+-dependent motility in alkaliphilic B. pseudo-
firmus OF4 to be MotPS, MotAB-like proteins.
Sodium Ion and Membrane Transport
As already described, alkaliphilic microorganisms grow
vigorously at pH 9–11 and require Na+ for growth. The
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Growth pH Ion requirement
No growth at pH 7 Na+ (essential)
Capable of growth at pH 7 Na+ (essential)
Capable of growth at pH 7 and 10 Na+ and K+ (essential)
presence of sodium ions in the surrounding environment
has proved to be essential for effective solute transport
through the membranes of alkaliphilic Bacillus spp.
According to the chemiosmotic theory, the proton-motive
force in the cells is generated by the electron transport chain
or by excreted H+ derived from adenosine triphosphate
(ATP) metabolism by ATPase (adenosine triphosphatase).
H+ is then reincorporated into the cells with cotransport of
various substrates. In the case of Na+-dependent transport
systems, the H+ is exchanged with Na+ by Na+/H+ anti-
porter systems, thus generating a sodium-motive force,
which drives substrates accompanied by Na+ into the
cells. The incorporation of a test substrate, a-aminoiso-
butyrate (AIB) increased 2-fold as the external pH shifted
from 7 to 9, and the presence of sodium ion significantly
enhanced the incorporation; 0.2 mol L21 NaCl produced
an optimum that was 20 times the rate observed in the
absence of NaCl. Other cations, including K+, Li+, NH4+,
Cs+ and Rb+ showed no effect, nor did their counteranions
(Krulwich, 1995). See also: Bacterial Cytoplasmic Mem-
brane; Bacterial Membrane Transport: Organization of
Membrane Activities
Mechanisms of Cytoplasmic pH
Regulation
The cells have two barriers to reduce pH values from 10.5 to
8 (Figure 3). (1) Cell walls containing acidic polymers func-
tion as a negatively charged matrix and may reduce the pH
value at the cell surface. The surface of the plasma mem-
brane must presumably be kept below pH 9, because the
plasma membrane is very unstable at alkaline pH values
much below the pH optimum for growth. (2) Plasma mem-
branes may also maintain pH homoeostasis by using Na+/
H+ antiporter system and ATPase-driven H+ expulsion.
Work in several laboratories on the critical antiporters in-
volved in this cycle has begun to clarify the number and
characteristics of the porters that support active mecha-
nisms of pH homoeostasis (Ito et al., 1997). As the result of
these mechanisms, the cell surface may play a key role in
keeping the intracellular pH value in the range between
7 and 8.5, allowing B. halodurans C-125 (and probably
other alkaliphiles) to thrive in alkaline environments.
See also: Bacterial Cytoplasmic Membrane; Bacterial
Intracellular Membranes

Extracellular (pH 10.5)
Cell wall Cell wall (peptidoglycan + acid polymers)
negatively charged
Periplasmic
space (pH 9) H+
H+
Cell
membrane 1 3
Intracellular (pH 8) 2
Na+ H+ Na+ H+
H+
1. Respratory chain 2. FoF1-ATPase
4. ∆ψ-dependent Na+/H+ antiporter
6. Oligopeptides/Na+ symporter
Figure 3 A schematic representation of cytoplasmic pH regulation.
The whole genome sequence of B. halodurans C-125 was
recently determined and an operon of poly-g-L-glutamate
synthetase genes was found. These results show that the
cells of B. halodurans C-125 are enclosed by at least two
acidic polymers such as teichuronic acid and poly-g-L-
glutamic acid with highly negative charges. Donnan equi-
librium in the bacterial cell wall was calculated to estimate
the pH values inside the polymer layer of the cell walls when
the outer aqueous solution is alkaline in nature. The fixed
charge concentration in the polymer layer was estimated to
be 2–5 mol L21 from the data reported for Gram-positive
bacteria, particularly for an alkaliphilic bacterium B. ha-
lodurans C-125. According to Tsujii’s calculation (Tsujii,
2002), the pH values estimated to exist inside a polymer
layer (cell wall) are more acidic than those of the surround-
ing environment by 1–1.5 U. Furthermore, whole genome
analysis of another alkaliphilic Bacillus strain, Oceanoba-
cillus iheyensis also exhibited that the putative protein
showed significant similarity to the tupA gene products of
B. halodurans C-125. Therefore, acidic polymers of poly-g-
L-glutamic acid and teichuronic acid are one of the im-
portant components in the cell wall of the alkaliphilic B.
halodurans and contribute to the regulation of pH ho-
moeostasis in the cytoplasm.
Genetics
Characteristics of alkali-sensitive mutants
Many alkali-sensitive mutants of alkaliphilic B. halodurans
C-125 have been obtained by chemical mutagenesis. This
strain grows well on Horikoshi minimal medium (supple-
mented with 0.2% (w/v) glutamate and 0.5% (w/v) glycerol
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Alkaliphiles
Flagellum
Na+
Amino acids Oligopeptides
Na+
H+ H+ Cell surface
+++++
4 5 6 7
----
Na+
Amino acids Na+ Oligopeptides
3. ∆pH-dependent Na+/H+ antiporter
5. Amino acids/Na+ symporter
7. Flagellar motor (mot)
as nutrients in place of glucose or starch, polypeptone and
yeast extract) over the pH range 7.5–10. One of the mu-
tants, 38154, was unable to sustain low internal pH in the
presence of Na2CO3, suggesting a defect in some aspect of
the regulation of internal pH. See also: Bacterial Genetics
A 5.1 kb parental DNA fragment from the parent strain
that restored the growth of mutant 38154 at alkaline pH
was isolated and cloned. The transformant was able to
maintain an intracellular pH that was lower than the ex-
ternal pH and contained an electrogenic Na+/H+ antipor-
ter driven only by Dc (membrane potential, interior
negative). Membrane vesicles prepared from mutant
38154 did not show membrane potential Dc-driven Na+/
H+ antiporter activity. These results indicate that the muta-
tion in 38154 affects, either directly or indirectly, elect-
rogenic Na+/H+ antiporter activity. This is the first report
of a DNA fragment responsible for a Na+/H+ antiporter
system in the mechanism of alkaliphily (Kudo et al., 1990;
Kitada et al., 1994). DNA fragments having the same DNA
sequences of pALK were also found in B. pseudofirmus
OF4, O. iheyensis, Bacillus clausii and B. subtilis 168.
Whole genome sequences of alkaliphilic
Bacillus strains
How alkaliphiles adapt to their alkaline environments is
one of the most interesting and challenging topics facing
microbiologists that might be clarified by genome analysis.
Two physical maps for alkaliphilic Bacillus strains have
been published: those for B. halodurans C-125 and Bacillus
firmus OF4 (Grønstad et al., 1998; Takami et al., 1999).
As shown in Figure 4, the 4 202 353 bp genome of
B. halodurans C-125 was determined in 2000. The genome
5
 Alkaliphiles
Teichuronic acid and
40 Mb 0 oriC
poly-γ-L-glutamic acid
responsible for Amylase
alkaliphily Cellulase
Pectinase
β-1,3-Glucanase
Protease
B. halodurans C-125 1.0
4202352 bp
3.0
pALK system
responsible for alkaliphily
2.0
Xylanse
Figure 4 Circular structure of Bacillus halodurans C-125 genome.
contains 4066 predicted protein-coding sequences, approxi-
mately 55% of which have functional assignments and the
rest are not clear yet. The genome contains 120 transpo-
sases, indicating that they have played an important evo-
lutionary role. There are clusters of poly-g-L-glutamate
synthesizing enzyme gene and pALK system gene that con-
tribute to alkaliphily of B. halodurans C-125. Many alkaline
enzyme genes are also detected, some of which have useful
industrial applications. Two years later, another whole
genome sequence of O. iheyensis HTE831 was deter-
mined, which is an alkaliphilic and extremely halotolerant,
isolated from a deep-sea sediment in the Mariana Trench.
The genome consists of 3.6 Mbp, encoding proteins such as
potentially associated with roles in regulation of intracel-
lular osmotic pressure and pH homoeostasis. Other several
open reading frames of Na+/H+ have been detected, and
further works are in progress. See also: Bacillus subtilis as a
Model for Bacterial Systems Biology
Alkaline Enzymes
Studies of alkaliphiles have led to the discovery of many
types of enzymes that exhibit interesting properties. The
first report concerning an alkaline enzyme published in
1971 described an alkaline protease produced by Bacillus
sp. 221. Strain 221 showed significant growth at pH 11 but
not at pH 7, and the alkaline protease had an optimal pH
for activity of pH 11.5–12, showing substantial activity
remaining at pH 13. In the author’s laboratory, more than
35 new enzymes have been isolated and purified. Some
of these have been produced on the industrial scale.
See also: Enzymes: General Properties
Alkaline proteases
An alkaliphilic Bacillus, Bacillus clausii 221 (ATCC 21522)
produces an alkaline protease that can be readily crystal-
lized because of the low content of contaminating proteins,
one of the advantages of alkaline enzymes (Horikoshi,
1971a). This enzyme possesses an optimal pH for activity of
6 ENCYCLOPEDIA OF LIFE SCIENCES & 2008, John Wiley & Sons, Ltd. www.els.net
11.5–12 and is stable at 608C for 10 min. The enzyme is also
stable at alkaline pH as high as 13. Moreover, the specific
activity is almost eight times higher than that of subtilisin.
Interestingly, the isoelectric point of the enzyme is also
highly alkaline (pH 11). One of the alkaline proteases pro-
duced by Bacillus halodurans AH-101 showed a high ac-
tivity against natural insoluble fibrous proteins such as
elastin and keratin. Attempts to improve the properties of
alkaline proteases for their use as detergent additives have
been going on for nearly 20 years and many alkaline pro-
teases are now employed in a number of detergent formu-
lations. In 1972, my colleague Yoshida isolated Bacillus
cohnii D-6 (FERM P-1592) and purified a very stable al-
kaline protease in the presence of detergents containing
high concentration of perborate in the absence of calcium
ion, although the enzyme production was too low. If the
enzyme productivity could be increased, the D-6 enzyme
would be the best enzyme for detergent additives. Almost
30 years later, Saeki et al. (2000) dramatically increased the
productivity to more than 20 g L21 by using gene engi-
neering technology. Recently, this old but new enzyme
has been added to one of the laundry detergents. See also:
Proteases
Alkaline amylases
Many alkaline amylases have been isolated from alkali-
philes. One of them, an alkaline amylase from an alkali-
philic Bacillus A-40-2 (ATCC 21592), has an optimal pH
for activity of 10–10.5 and also shows 50% of the activity
remaining at pH 9 and 11.5. This enzyme hydrolyses about
70% w/w of starch in the manner of an a-amylase (Ho-
rikoshi, 1971b; Yamamoto et al., 1972). Recently, Ca2+-
free amylase, AmyK38 requiring Na+ for its activity was
discovered from an alkaliphilic Bacillus sp. KSM-K38 that
is highly resistant to chelating reagents and chemical ox-
idants. A haloalkaliphilic archaeon, Natronococcus sp.
strain Ah-36, produced an extracellular amylase. The am-
ylase exhibited maximal activity at pH 8.7 and 558C in the
presence of 2.5 mol L21 NaCl. Maltotriose is actually the
digestion product from soluble starch. The gene has been
cloned and expressed in another halophilic archaeon
Haloferax volcanii. However, no industrial application
has been found to date.
Cyclodextrin glucanotransferase
In 1969, Corn Products International Co., USA, produced
b-cyclodextrin (b-CD) using Bacillus macerans cyclodex-
trin glucanotransferase (CGTase). Teijin Ltd. in Japan also
produced b-CD using the Paenibacillus macerans enzyme
in a pilot plant. There were several serious problems in both
production processes, including (1) the yield of CD from
starch was not high and (2) toxic organic solvents such
as trichloroethylene, bromobenzene and toluene were
required to precipitate CD because of the low yield.
Alkaliphilic Bacillus 38-2 (ATCC 21 783) produces a
CGTase with excellent properties. Although this enzyme

is not an alkaline enzyme in the strict sense, it is stable up to
pH 10. The optimum pH for the activity is significantly
broad, ranging from pH 4.5 to 10. In addition, the conver-
sion ratio from starch to cyclodextrin reaches 75–80%,
exceeding that of the conventional enzyme from B. mace-
rans, which achieves only 50%. These high yields enabled
direct crystallization of b-CD from the primary enzyme
reaction mixture at low cost without solvent precipitation,
facilitating industrial production. This has paved the way
for its use in large quantities in foodstuffs, chemicals and
pharmaceuticals. This finding has stimulated the research
for and discovery of many cyclodextrin-forming enzymes
from alkaliphiles.
Pullulanases
An alkaline pullulanase of Bacillus sp. 202-1 was discov-
ered in 1975. The enzyme has an optimum pH at 8.5–9 and
is stable for 24 h at pH 6.5–11.0 and 48C. The enzyme is
most active at 558C, and is stable at up to 508C for 15 min in
the absence of substrate. In the course of screening alkaline
cellulases for detergent additives, a novel alkaline pull-
ulanase from alkaliphilic Bacillus sp. KSM-1876 having an
optimum pH for enzyme action of around 10–10.5 was
isolated. This enzyme is a good candidate for use as an
additive to dish-washing detergents. However, no indus-
trial application has been developed to date.
Alkaline Cellulases
Alkaline cellulases (carboxymethylcellulases, CMCases)
that degrade substrates at pH 9–10 are produced by a
number of alkaliphilic Bacillus strains. Alkaliphilic Bacillus
cellulosilyticus N-4 produces several of these cellulases
(Horikoshi et al., 1984). Crude cellulase preparations ex-
hibit a broad pH range (4.5–11) for optimal activity. These
complex enzymes have been analysed by cloning of the
genes encoding for the constituent enzymes in E. coli. An-
other alkaliphile, Bacillus akibai 1139 produces an alkaline
cellulase that has an optimum pH for activity of 9 but that is
not active at pH 7. The discovery of alkaline cellulases
promoted the development of a new industrial application
of cellulases as laundry detergent additives. See also:
Cellulose: Biogenesis and Biodegradation
Many alkaline cellulases used as laundry detergent ad-
ditives have subsequently been isolated and marketed.
Yields of enzyme can be 20–30 g from 1 L of culture broth,
some of the highest yields for enzymes so far reported.
Xylanases
The first article reporting a xylanase from alkaliphilic Ba-
cillus sp. C-59-2 was published in 1973. The purified enzyme
exhibited a broad optimum pH range for activity of 6–8.
B. halodurans C-125 was subsequently shown to pro-
duce two xylanases. Xylanase A had a molecular mass of
43 000 Da, whereas that of xylanase N was 16 000. Xylanase
ENCYCLOPEDIA OF LIFE SCIENCES & 2008, John Wiley & Sons, Ltd. www.els.net
Alkaliphiles
N was most active at pH 6–7 and xylanase A was most
active at pH 6–10, with some activity at pH 12. Both
xylanases had trans-xylosidation activities. Xylanase A be-
longs to the family F group of xylanases.
After the demonstration that alkali-treated woodpulp
could be biologically bleached by xylanases instead of by
the usual environmentally damaging chemical process us-
ing chlorine, the search for thermostable alkaline xylanases
has been extensive. For example, alkaliphilic and therm-
ophilic Bacillus sp. strain TAR-1, isolated from garden soil,
produced a xylanase extracellularly. The xylanase was
most active over a pH range 5–9.5 at 508C. Optimum tem-
peratures of the crude xylanase preparation were 758C at
pH 7 and 708C at pH 9. An alkalitolerant Cephalosporium
(NCL 87.11.9) has been isolated from soil samples that is
capable of rapid growth and xylanase secretion over a wide
pH range (pH 4–10). Thermophilic and strictly anaerobic
xylanase-producing Dictyoglomus strains were isolated
from different environments: a strain of Dictyoglomus
thermophilum from a hot spring in Japan; strain B1 from a
paper-pulp factory in Finland; and strain B4a from a ther-
mal pool in Iceland. The xylanases of all the isolates had a
broad pH range of activity (5.5–9) with optimum temper-
atures at 80–908C. These xylanases were inactive against
cellulose, including crystalline cellulose, indicating a pos-
sible application of these enzymes in biological bleaching
processes.
Pectinases
An alkaline endo-polygalacturonase produced by alkali-
philic Bacillus sp. P-4-N was discovered in 1972. The op-
timum pH for enzyme action was 10 for pectic acid. Since
then, several other endo-polygalacturonate lyase produc-
ing alkaliphilic microorganisms such as Bacillus sp. RK9,
B. halodurans C-125, Bacillus sp. strain KSM-P15 etc. have
been isolated. The optimum pH for the enzyme activity
towards acid-soluble pectic acid was 10. Three articles on
the potential applications of alkaline pectinases have been
published. The first application of alkaline pectinase-pro-
ducing bacteria was in retting during the Mitsumata bast
paper process. The pectic lyase (pH optimum 9.5) produced
by an alkaliphilic Bacillus sp. GIR 277 has also been used in
improving the production of another type of Japanese pa-
per, the new retting process producing a high-quality, non-
woody paper that was stronger than the paper produced by
the conventional method. Hoondal et al. (2002) recently
reviewed the microbial alkaline pectinases and their po-
tential industrial applications.
Other enzymes
Several other enzymes from alkaliphilic or alkalitolerant
organisms have been studied but have not yet found com-
mercial application. Alkaline b-1,3-glucanases, which are
active at pH greater than 8, were purified from the culture
broths of alkaliphilic Bacillus sp. K-12-5 and Bacillus sp.
221 isolated from soil. Many alkaline lipases have been
7
 Alkaliphiles
isolated from a variety of different alkaliphiles. These en-
zymes will also be used as enzyme additives to detergents in
the near future.
Future of Alkaliphiles
Since the rediscovery of alkaliphilic bacteria by the author,
more than 1500 articles have been published on many
aspects of alkaliphiles and alkaliphily. The alkaliphiles are
unique microorganisms with great potential for micro-
biology and biotechnological exploitation. Aspects that
have received the most attention in recent years include:
(1) extracellular enzymes and their genetic analysis;
(2) mechanisms of membrane transport and pH regula-
tion and (3) the taxonomy of alkaliphilic microorganisms.
It is generally accepted that less than 5% of all existing
microorganisms have been obtained in pure culture and
tested for their microbial properties. The remaining 95% or
more have either not been discovered or are impossible to
grow reproducibly in culture. Fortunately, the biotechno-
logical potential of alkaliphiles made exponentially
increase the isolation and characterization of new organ-
isms. As the results of these works, physiology, molecular
biology and genetics of alkaliphiles have been developed
quickly.
Several alkaliphiles are extensively studied on alkaliphily
by using genomic approaches that are very useful methods
to understand these organisms. What will be the next line of
development? Beside this, we should investigate how to
isolate and cultivate so-called ‘uncultured microorganisms’
as living creatures, although somewhat boring and time-
wasting experiments. Fusion of in silico and wet research
works should make great progress in biology of alkaliphiles
after long confusion of biologists.
I would like to say as follows:
Microorganisms can do anything you want, if you know
how to ask them.
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Further Reading
Grant WD, Mwatha WE and Jones BE (1990) Alkaliphiles:
ecology, diversity and applications. FEMS Microbiology
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Horikoshi K (1999) Alkaliphiles. Tokyo: Kodansha.
Horikoshi K (2006) Alkaliphiles: Genetic Properties and Applica-
tions of Enzymes. Tokyo: Kodansha.
Horikoshi K and Akiba T (1982) Alkalophilic Microorganisms: A
New Microbial World. Heidelberg: Springer.
Horikoshi K and Grant WD (eds) (1991) Superbugs. Heidelberg:
Springer.

Horikoshi K and Grant WD (eds) (1998) Extremophiles: Micro-
bial Life in Extreme Environments. New York: Wiley-Liss.
Jones BE, Grant WD, Duckworth AW and Owenson GG (1998)
Microbial diversity of soda lakes. Extremophiles 2: 191–200.
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acid uptake in membrane vesicles of alkalophilic Bacillus no.
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Kroll RG (1990) Alkaliphiles. In: Edwards C (ed.) Microbiology
of Extreme Environments, pp. 55–92. New York: McGraw-Hill.
ENCYCLOPEDIA OF LIFE SCIENCES & 2008, John Wiley & Sons, Ltd. www.els.net
Alkaliphiles
Krulwich TA and Guffanti AA (1989) Alkalophilic bacteria.
Annual Review of Microbiology 43: 435–463.
Krulwich T, Ito M, Gilmour R and Guffanti A (1997) Mecha-
nisms of cytoplasmic pH regulation in alkaliphilic strains of
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Krulwich T, Ito M, Hicks DR, Gilmour R and Guffanti A (1998)
pH Homeostasis and ATP synthesis: studies of two processes
that necessitate inward proton translocation in extremely alka-
liphilic Bacillus species. Extremophiles 2: 217–222.
9