FEMS MicrobiologyReviews75 (1990) 255-270 255

Published by Elsevier

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FEMSRE 00143

AlkaliphHes
Alkaliphiles: ecology, diversity and applications
W . D . G r a n t 1, W.E. M w a t h a 2 and B.E. Jones 3

I Departmem of Microbiology, University of Leicester, Leicester, U.K., z Kenyatta Uniuers~ty, NairobJ, Kenya,
and 3 GisJ-Brocades Research and Development. Delft. The Netherlands

Key words: Alkaliphilic bacteria; Ecology; Enzyme application

1. SUMMARY in certain parts of the world. Alkaliphilic micro-

Organisms with pH optima for growth in excess
of prig, usually between 9 and 10, are properly
defined as alkaliphiles (or sometimes alkalophiles).
Most of the organisms described to date as grow-
ing under very alkaline conditions are pro-
karyotes, comprising a heterogeneous collection of
eubaeteria (including cyanobacteria) with a few
examples of archacbactesia. With the exception of
the alkaliphilic archaebacteria (which are also
halophilic), few of the isolates have been compre-
hensively classified. However, it is clear that many
different taxa are represented amongst the al-
kaliphiles, and that some of these at least are
likely to be new taxa. Alkaliphiles can be isolated
from 'normal' environments such as garden soil
presumably because there are transient alkaline
conditions generated in such environments by bio-
logical activity, but a relatively restricted range of
types results from the exploration of such environ-
ments (often Bacillus spp.). Naturally-occurring
stable alkaline environments such as eutrophic
soda (Na2CO3) lakes harbour a much wider range
of types. A similarly wide range of types may exist
in oligotrophic Ca(OH)z-dominated ground water
Correspondence to: W.D. Grant, Department of Microbiology,
University of Leicester, UniversityRoad, LeicesterLEt 7RH,
U.K.
organisms have already made a large impact in the
application of bioteehnology for the manufacture
of mass market consumer products. " Biological
detergents" contain enzymes that have usually
been obtained from alkaliphilie or alkalitulerant
bacteria. The current proportion of total world
enzyme production destined for the laundry deter-
gents market comfortably exceeds 25%, and there
are other possible applications in food and wasl~
treatment industries. To date, most commercially
available enzymes are derived from a few bacterial
taxa (notably Bacillus spp.) and we have been very
conservative in our choice of environments in
which to look for new products. It is reasonable to
suppose that soda lakes (and other alkaline en-
vironments) wiU yield a wealth of new organisms
with useful commercial properties.
2. ECOLOGY
Alkaliphilic microorganisms (particularly pro-
karyotes) are widely distributed and can be found
in almost any environment, even in environments
where the overall pH may not bt~ particularly
alkaline. Organisms with pH optima for growth
> pH8, usually between 9 and 10, are defined as
alkaliphiles. Obligate alkaliphilcs are incapable of
growth around neutrality. The widespread pres-

0168-6445/90/S03.50 © 1990 Federation of Europe.an MiczobiologiealSocieties

256
ence of alkaliphiles in, for example, neutral or
even acid soils indicates that transient alkalinity
arises through biological activity such as ammoni-
fication, sulphate reduction, or photosynthesis [1].
Commercial processes such as cement manufac-
ture (and casting), elcetroplating, paper manufac-
ture and the lye treatment of potatoes and animal
hides also generate alkaline conditions. It is to be
supposed that these processes produce wastes ca-
pable of supporting the growth of particular al-
kaliphiles, although tittle or no microbiological
information is available for most of these environ-
merits. One exception is the alkaline process for
removing potato skins, where an unusual non-
spore forming Gram-positive bacterium exten-
sively colonizes the waste [3,4].
The most stable, naturally-occurring alkaline
environments are caused by a combination of
geological, geographical and climatic conditions,
and a far more diverse population of alkaliphiles
is to be found in such habitats.
2.1. Low Ca 2+ environments (soda lakes and de-
serts)
Alkaline soda lakes and soda deserts are widely
distributed, although little explored, probably be-
cause of inaccessibility in most cases. Lists of such
lakes and deserts together with geographical loca-
tions are given in Grant and Tindall [5] and Tindall
[6]. These environments are characterized by the
presence of large amounts of sodium carbonate
(or complexes of this salt) formed by evaporative
concentration. In the course of the formation of
alkalinity, other salts (particularly NaC1) also con-
centrate, generally giving rise to environments that
are both alkaline and somewhat saline.
There are a number of theories concerning the
source of the soda. The simplest theory supposes
an unusual geology where the rooks already con-
rain large amounts of soda. For example, the East
African Rift Valley, an area of active vulcanism.
contains many soda lakes, and at least one active
volcano contributes a source of sodium carbonate
to the surface environment [7]. However, it is
unlikely that this is the explanation for the genesis
of alkalinity in most of these lakes. The likely
mechanisms contributing to the formation of al-
kalinity have been reviewed by a number of re-
searchers [8-11]. It is clear that particular geologi-
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cal and climatic features give rise to alkalinity,
particularly the absence of significant amounts of
Ca 2+ and Mg 2+ in the surrounding topography.
In surface and near surface zones, weathering
and biological activity produces CO2-charged
surface waters and thus a bicarbonate-carbonate
solution is produced which leacbes surrounding
minerals, which in turn reflect the surrounding
geology. Concentration of ions by evaporative
concentration leads to a shift in the carbon di-
oxide/bicarbonate/carbonate equilibrium. In
most environments such groundwaters rapidly be-
come saturated with respect m Ca 2+, resulting in
the precipitation of calcite (CaCO3), often in as-
sociation with magnesite ~MgCO3) and dolomite
(MgCa(CO3)2). Thus in situations where the con-
centration of Ca 2+ and M g 2+ exceed that of
CO 2 - , carbonate is removed from solution and
the genesis of an alkaline brine is inhibited (as is
the case for most groundwaters). If the CO 2-
concentration exceeds that of Ca 2+ and Mg 2÷,
alkalinity develops, usually with N a + as the domi-
nant cation.
The East African Rift Valley is a typical low-
Ca 2+ area, characterized by a geology dominated
by alkaline trachyte lavas (high N a ÷, low M g 2+,
Ca 2+) [7,12,13] in a number of distinct isolated
drainage basins, the majority of which are closed.
Consequently, a considerable number of soda lakes
have developed along the floor of the valley. Fig. 1
iilustf~,t~ the schematic representation of the pro-
posed mechanisms involved in the formation of
Lake Magadi, an alkaline and saline lake some
200 km south of Nairobi at the lowest point in the
Rift Valley. However, it is clear that subterranean
flows which may arise in deeper, non-alkali rich
strata may contribute to the final ionic composi-
tion of such lakes, and may account for occasional
fresh water lakes in close proximity to soda lakes
(e.g. Lake Nalvasha, Kenya). Bacterial sulphate
reduction in surrounding swamps has also been
proposed to contribute towards alkalinity in cer-
tain soda lakes such as those in the Wadi Natrun
depression and certain Hungarian soda lakes
[14,15] but is unlikely to be a general mechanism.
Undoubtedly, the best studied soda lakes are
those of the East African Rift Valley, where de-
 257
WEST EDGE OF RIFT EAST EDGE OF RIFT

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RIFT VALLEY

<C ' ; , , ~,~, CONCENTRATION Na* K ÷

i ~-- c~-
~ Ca CaCO3

DEEP ~"GROUNDWATER ~ RESERVOIR ~.~,_~

Fig. I. Schematicrepresentation of the pos.~iblernechanisn~ involvedin the function of alkaline saline lakes (L. Magadi)(modified
from1271).

tailed limnological comparisons with other Afri-
can lakes have been carried out over the years
[5,16-23]. Detailed analyses of the soda lakes of
America are available from a variety of sources
[10,24], and the l a k ~ of the Wadi Natrun depres-
sion have also been examined in detail [25]. Rather
less information is available about Hungarian soda
lakes [15] and the lakes of the Kulunda Steppe
[26]. Tindall [6] lists a number of references relat-
ing to analyses of Australian and Antarctic lakes
which may be alkaline and saline.
The African Rift Valley is probably unusual in
having lakes with significant, largely permanent
bodies of brine, and a range of total salinities
from around 5~o ( w / v ) in the more dilute lakes
(e.g. Boeotia, Elmenteita, Nakuru etc.) to satura-
tion (30~o or greater) in parts of lakes Magadi and
Natron. All of the soda lakes in the Kenyan-
Tanzanian part of the Rift Valley are devoid of
significant amounts of Ca 2+ and Mg 2÷ (in most
eases bolow the level of detection), pH values
range from 10 to 11.5 (the most concentrated lakes
have the highest pH values) with total alkalinity
ranging from < 100 mM to > 2000 raM, depend-
ing on total salinity. Na2CO 3 and NaCI are the
main salts present in a ratio of approximately 2 : 1
respectively, with K + the only other ion present in
significant amount (5-200 mM depending on total
salinity). Grant and Tindall [5] and Eugster and
Hardie [10] provide detailed chemical analyses on
a range of East African Lakes. One of the features
of these lakes is that the total salinities may vary
considerably according to the seasonal weather
conditions, although the pH remains more or less
constant.
2.2. High Ca 2÷ environments
Highly alkaline Ca 2 +-hearing groundwaters are
extremely rare. However, such groundwaters where
highly alkaline conditions persist ( > pH 11) have
been identified in various geological locations in
California [28], Oman, Yugoslavia [29], Cyprus
[30] and Jordan [31]. Several groundwater springs
in Oman have been subjected to a preliminary
microbiological analysis [32]. This type of alkaline
groundwater is of interest as a model system,
because the hydrochemical characteristics resem-
ble the inferred conditions in cement pore waters.
Concrete is a major structural component in many
long-term storage repositories (including those
258
M g F o S I O 4 * C 0 2 * H20
OLIVINE
MgCaFeSiO 3 * CO2 * H20 "~-~"
PYROXENE
Mg 2 * * H 4 S I O 4 * H 2 O
MgFoSIO 4 + MgCaFeSIO 3 * H20
Fe(OH) 2
Fig. 2, Chemical reactions involved in the generation of alkalinity in the Oman groundwaters.
proposed for the containment of nuclear waste),
and thus there is a need to assess any microbial or
chemical activity that might compromise the
long-term integrity of soch structures.
The chemistry of such alkaline groundwaters is
determined by the low-temperature weathering of
two l:,'imary minerals in the host rock, olivine and
pyroxene. In near surface zones, CO2-charged
surface waters decompose these minerals in the
way depicted in Fig. 2, resulting in Ca 2+ and
O H - being released into solution. This process
inevitably leads to calcite precipitation initially,
but unlike the situation existing in soda lake areas,
carbonate quickly becomes depleted, after which a
Ca(OH)2 dominated brine is produced, where solid
(Ca(OH) 2 is in equilibrium with the soluble phase,
maintaining a highly alkaline environment ( > pH
11). Na + and CI- are introduced by leaching of
entrapped mineral salts. Highly reducing condi-
tions are produced by the release of Fe 2+. Hydro-
gen gas is also evolved by the oxidation of H20 by
transient metal hydroxides. As such groundwaters
reach the surface, calcite may precipitate on con-
tact with atmospheric CO2, leading to some deple-
tion of Ca 2÷. Mg 2+ is removed as serpentine and
by precipitation as magncsite and brucite. Bath et
al. [32] report that at the sites sampled west of
Muscat on the Gulf of Oman, the major ions were
CI-, Na +, O H - , Ca 2÷ and K ~', in decreasing
order of amount, and that carbon, nitrogen and
phosphorous contents were in the range seen in
ultra-oligotrophic waters, pH values ranged from
~ M g 2+ + H C O ~ + H 4 S I O 4 ÷ Fe 2 . • O H -
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Mg 2~ * Ca 2+ * H C O ~ + H 4 S I O 4 * Fe 2 . * OH-
Mg3Si2Os(OH) 4 * H÷
SERPENTINE
~- M g 3 5 i 2 O s ( O H ) 4 + Fe 2+ * Ca 2 . * O H -
*z- Fe203 . H2 + H20
11.16 to 11.44. Thus a dilute chemistry (major ions
present at 10 mM) quite unlike tiaat of the soda
lake (major ions present molar concentrations) is
generated, where the alkalinity is due to O H - in
the absence of CO~-. In addition, conditions are
profoundly reducing in most cases [32 I.
3. DIVERSITY
3.1. H i g h - C a 2 + e n u i r o n m e n t s
Analyses carried out by Bath et al. [32] indicate
a low bacterial population in the Oman springs.
Viable counts of organotrophs were between 10 I
to 10 4 cfu ml -t. Most of the isolates were faculta-
tively anaerobic, reflecting the reducing conditions
in the environment, but despite most strains grow-
ing at pH 10 or above, only a few isolates were
obligately alkaliphilic. Enrichments for nitrifying,
denitrifying, sulphur-oxidizing and methanogenic
bacteria were negative. However, unidentified
sulphate-reducing bacteria proliferated in enrich-
ments at high pH. Since the samples were taken
from seepage.s to the surface, both oxygenic and
anoxygenic photolrophic bacteria were isolated in
enrichments.
Attempts were made to identify the organo-
trophie bacteria using commercially available sys-
tems and a variety of morphological and biochem-
ical tests. Bath et al. [32] came to the conclusion
that the microbial genera found in these alkaline
springs were similar to those in less extreme soil
 259

Table ] bacterial blooms [5], but may also be orange {36]

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Tentativeid~ntifir.ationof isolatesfromspringwaters in Oman or red [5,37] due to other organisms.
1321 This is reflected in the extremely high primary
Genus Number of isolates productivities associated with some of these lakes.
Actinobaciltus 4 The mean gross primary productivity for the
Bacillus 4 streams and lakes of the world derived by Whit-
Hafnia 3 taker and Likens [38] is about 0.6 g C m -2 day -1.
Vibrio 2 Soda lakes often exhibit productivity rates > 10 g
Clostridium 2 C m- 2 day- i [18,19]. Such rates are normally only
Caulobacter t
Serratia l
encountered in lakes enriched by human enter-
Enterobactefiaccae 2 prises, and accordingly, soda lakes are probably
Flavobacterium 2 the most productive, naturally occurring aquatic
Hyphomicrobium 1 environments in the world, presumably due to the
Pseudomonas l virtually unlimited reserves of CO 2 available for
Coryneform 1
Unknown 4 autotrophie growth in these carbonate-laden
waters. The lakes of the East African Rift Valley
form a good example of the type, and most of
what follows relates to this geographical location.
and water environments. At least some of the The primary productivity is generally due to
bacteria could be attributed to contamination of the presence of dense populations of cyano-
the spring waters by animai~. Table 1 lists genera bacteria (up to 13000 cyanobacterial filaments
isolated from the springs. It is of note that the ml -I) [18,19]. In extreme cases, the blooms may
organisms were, in general, quite versatile, utiliz- consist almost entirely of one species, usually
ing a wide range of sugars and significant num- Spirulina sp. These particular cyanobaeteria are
bers were gelatinase and amylase positive. the principal food of the immense flocks of
It is likely that the alkaline spring investigated flamingoes that inhabit the Rift Valley. The
by Souza and Deal [33] that yielded a eoryneform larger-celled species are gas-vacuolate [5], accu-
species had a similar genesis and chemistry to the mulating along the shore lines of the less saline
Oman springs. It is not clear whether any of these lakes as thick scums. However, aerial views indi-
isolates actually grow at pH values of more than cate similar densities throughout some of the lakes.
11 or so, because of the technical difficulties of These uyanobacteria, however, do not normally
maintaining media of very high pH [34,35]. Nor is bloom in the more saline lakes such as Lakes
it clear if the organisms are adapted to grow Magadi and Natron, unless a rare prolonged rainy
rapidly within the spring waters rather than being spell results in a significant reduction in the con-
adapted for survival under these conditions. Pre- ductivity of the brines. Spirulina spp. are of con-
liminary results using enrichments with cement siderable interest as protein sources for a variety
factory effluents kept at pH 11.5-12.0 by contact of uses and a number of semi-commercial oper-
with solid phase Ca(OH)2 suggest that growth ations seek to exploit the valuable biomass pro-
under these conditions is uncommon (W.D. Grant, duced in various soda lakes [39l.
unpublished results). Other cyanobacteria commonly participating in
bloom formation include heterocystous fila-
3.2. L o w Ca 2 . environments (soda lakes a n d soda mentous Anabaenopsis spp., recently reclassified
deserts) as Cyanospira spp. [40], Unicellular cyanobaeteria
One of the most striking features of soda lakes which may be Synechococcus or Chroococcus spp.
is the predominance of microorganisms, particu- (Gleocapsa?) [41] are also common and may occa-
larly phototrophs, as permanent or seasonal sionally dominate particular la,~es (e.g. Crater Lake
blooms. Thus, dependent on the water chemistry, west of Lake Naivasha). Eukaryote algae, such as
such lakes are likely to be green due to cyano- examples of the diatom genera Nitzsehia and
260
Navicula are also common and may comprise a
significant proportion of the phototroph popula-
tion and also occasionally dominate the environ-
ment [20], but generally at lower salinities.
There is also a substantial, but as yet unquanti-
fled contribution to primary productivity made by
anoxygenic phototrophic bacteria of the genus
Ectothiorhodospira [51. Aerial surveillance indi-
cates that significant blooms of these organisms
occur in Kenyan soda lakes from time to time
(often associated with cyanobacterial blooms in
other parts of the lake), and halophilic types are
found in the lakes of the Wadi Natrun depression
[25] and probably in western American soda lakes
[42]. These bacteria, although anaerobic in their
mode of growth, are relatively oxygen tolerant,
and presumably proliferate together with cyano-
bacteria, because these shallow highly eutrophic
lakes generate anoxic conditions in and close to
sediments as a consequence of organotrophic ac-
tivities. F,ctorhiorhodospira spp., which constitute
a separate and distinct lineage of phototrophic
bacteria, also participate in the sulphur cycle [43]
oxidizing H2S (presumably produced by as yet
uncharacterized alkaliphilic sulphate-reducing
bacteria), so their importanee in these lakes is
likely to be considerable.
The highly saline, alkaline brines of Lake Mag-
adi and the lakes of the Wadi Natrun have a
population of prokaryotes distinct from those in
the more dilute lakes. These very saline lakes are
eoloured red by large numbers (106-10 ~ ml - I ) of
haloalkaliphilic arehaebacteria [5,25]. To date,
these are the only known examples of obligately
halophilic and alkaliphilic microorganisms. RNA
sequence comparisons and nucleic acid hybridiza-
tion studies indicate that these organisms repre-
sent distinct evolutionary lineages within the gen-
eral archaehacterial halophile (halobacterial) line
[44,45]. All of the isolates examined to date have
rather similar physiological properties with pH
optima for growth between 9 and 10, salinity
optima between 2.5 and 4.0 M NaC1 [46], and
growth at very low Mg 2+ concentrations. Two
genera are currently recognized, Natronobacterium
(with three species) and the monospecifie Natro-
nococcus ( N. occultus ) [47,48]. Natronobacteria are
widely distributed in soda lakes, and like other
haiobacteria have red carotenoid pigments, mass
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developments imparting colouration to the en-
vironment [45]. Organisms of this type have been
described in the Wadi Natrun [25], American soda
lakes [49] and recently have been isolated from a
Tibetan soda Lake (W.D. Grant and H.W. Jan-
nasch, unpublished reports). Natronococci, on the
other hand, have only rarely been isolated from
alkaline brines [48], and microscopic examination
of brines indicates only rare occurrences of this
group.
Natronobacteria and natronococci are unusual
in that they, unlike other archaebacteria, have
membranes that contain significant amounts of
C25 (sesterterpanyl) isopranoid chains in ether lin-
kage to glycerol [50,51]. Detailed characterizations
of their complex lipids by N M R analyses [52] have
shows that these archaebacteria have a very simple
pattern of complex lipids composed exclusively of
phospholipids, with phosphatidylglyeerol (PG) and
phosphatidylglycerol phosphate (PGP) comprising
most of the complex lipid in all types. However,
there are trace amounts of other phospholipids
[49] that are species specific, and one of these has
recently been characterized as an unusual cyclic
phosphate derivative of PGP [53].
Natronobacterial blooms represent secondary
productivity in these lakes. The primary produc-
tivity is probably due to Ectothiorhodospira spp.,
although this is not proven (or possibly rare
cyanobacterial blooms occurring after heavy rain).
There is no doubt that the haloalkaliphilic archae-
bacteria are not in themselves photoautotrophie
since they, unusually amongst halobacteria, lack
the light-mediated bacteriorhodopsin proton pump
[54] which is coupled to energy generation in other
halobacteria, although they do possess other pho-
reactive retinal pigments.
Other archaebacteria are to be found in al-
kaline environments since certain soda lakes at
least show methanogenesis. A number of mildly
alkaliphilic and presumably halotolerant methano-
gens have now been isolated from the Wadi
Natrun, Lake Magadi and Big Soda Lake, Nevada
[55]. None of these isolates are as alkaliphilic and
halophilie as the natronobacteria and natrono-
cocci, but they indicate that it is likely that the
range of physiologies found in the "neutrophilic'
 261

:nvironment are likely to exist in the alkaliphilic results of the first 5 months (including one rainy

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:and haloalkaliphilic) environment.
So far, rather little attention has been paid to
the organotrophic eubacteria in soda lakes, al-
though these are readily isolated, using media
described by Horikoshi and Akiba [2] and G r a n t
and Tindail [34] at N a C I levels appropriate for the
particular lake under study. The remarkable
primary productivity supports a dense population
of bacteria.
T o t a l counts indicate popu]ations of 1 0 7 - 1 0 ~
bacteria m 1 - 1 [56], and viable counts (W.E.
M w a t h a and W.D. Grant, unpublished results) of
aerobic "copiotrophs' give 105-106 cfu m l - i on a
range of dilute soda lakes. We have recently begun
a systematic survey of the Kenyan Lake Bogoria,
Lake EImenteita and Lake Nakuru, monitoring
bacterial numbers and types throughout the year
(which usually has two rainy seasons), in relation
to conductivity, algal numbers and so on. T h e
season) indicate a stable population of around 10 5
cfu m l - i in all of the lakes, dominated by a few
types (Table 2), despite o f considerable fluctua-
tions in conductivity, particularly in Lake N a k a r u .
Furthermore, each lake experienced a major
cyanobacterial bloom dominated by Spiruflna spp.,
although Lakes EImenteita and N a k u r u had sig-
nificant numbers of other cyanobacteria. Similar
numbers of diatoms were found in all lakes
throughout the survey period, but as the cyano-
bacterial blooms decrease, the relative proportions
of these increase, despite a significant drop in the
total phototrophic biomass as measured by chlo-
rophyll a. T h e chlorophyll a values are broadly in
line with measurements m a d e over the years [17].
Despite the increeses in cyanobacterial biomass,
there is no evidence over this part of the survey of
any greater secondary productivity concomitant
with or following the cyanobacterial bloom.

Table 2
Analyses of Kenyan soda lakes
Month Lake Conduclivily Viable count Chlorophyll a Types of algae
(pScm i) bacteria (pgl i)
October 1988 Bogoria 41000 3.7 × 105 86 Spirulina, 85%; Nauiculo, Nitzschia
Elmcnleita 13000 3.0 ×10 ~ 40 Spirulina, 38%; Anabaenopsis, 27%;
Chro~occus, 10%
Nakuru 19000 2.9 × l0 s 53 Spirulina, 51%; Anabaenopsls. 30%;
Chroococcus. 18%
November 1988 Bogoria 40000 3.5 X l0 s 80 Spirulina, 95%; Nilzschia
EImenleita 13000 5.1 × 105 64 Spirulina, 51%; Anabaenopsis, 32%;
Chroococcus, 6~
Nakura I90G0 1.4 × l0 ~ 65 Spirulina. 68~; Anabaenopsis, 21%;
Chroococcus. 7~
December 1988 Bogori,L 39000 6.4 × lO~ 115 Spirulina, 73%; Oocystis, ?~avicula
Elrnenteila lb0~0 1.2 x 10~ 181 Spirufina, 31%; dnabaenopsi$. 27~;
Chroococcus, 23%
Nakuru 17000 2.3 × 106 83 Spirulina, 68%; Anabaemopsis, 23%;
Chroococcu~, 8%
January 1989 Bogoria 41 (300 3.0 X 105 86 Spirulina, 75~; Oocysti$, Nauicu(a
Elmenteita 16000 1.5 × 105 46 Spirulina. 35%: Anakoenopsis, 18%;
Clu'o~-occus, 23%
Nakuru 16000 1.75× 106 45 Spjrulina, 50%; Anabaenopsis, 25%;
Cbroococcus, 25%
I~eerua.,T 1 9 8 9 Bogaria 41000 1.4 × 105 13 Spirufina. 72~; Oocystis. Navicula
Elmenleita 19000 2.4 X 103 8 Chrc~coccus, 20~: Nitzschia. 40f~
Nakuru 30000 2.fi X 10~ 2 Spirufina, 50~; Anabaenop$i$. 15%;
Navicula, 30%
262
Many isolates of organotrophic bacteria have
now been obtained from these lakes and, as might
be expected, most are biochemically reactive pre-
sumably reflecting the nutrient-rich environment
that they inhabit. Cell wall analyses indicate that
the isolates are equally divided between Gram-
positive and Gram-negative types. Unlike soil en-
vironments, spore forming isolates are rare. The
percentage of G + C contents of the isolates cover
a wide range from 40-70%, indicating a wide
range of taxa. Certain of the isolates are common
to all the lakes, and the same types predominate in
any one lake over the period examined to date. As
might be expected, most of the isolates are oh-
ligately alkaliphilic. Preliminary evidence indicates
a comparable range of strictly anaerobic types.
The very saline lakes seldom yield other than
haloalkaliphilic archaebaeteria, but alkaliphilic
and halotolerant Bacillus types are occasionally
enriched ([57] and unpublished results).
It is clear that considerable work will be neces-
sary before the systematics of these soda lake
bacteria becomes clearer. Chemotaxonomic and
phylogenctic analyses will be central to their cir-
cumscription. Without robust systematics, only a
superficial analysis of the microbial ecology of
these odd environments will be possible.
4. APPLICATIONS
It is certainly true to say that alkaliphilic mi-
croorganisms have already made a large impact in
the application of biotechnology for the manufac-
ture of mass market consumer products. The so-
called "biological detergents" are one of the few
products of biotechnology industry used by the
ordinary consumer on an almost daily basis. These
detergents contain enzymes which often have been
obtained from alkaliphilic or alkalitolerant bacte-
ria. The importance of these products can be
appreciated when it is realised that in 1983 about
a quarter of the total world enzyme production
was destined for the laundry detergents market
[58l; and that currently the proportion is much
higher. The worldwide market is currently esti-
mated to be worth $200 million and the U.S.
market is forecast to more than double in value
been 1987 and 1995, rising from $75 million to
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$160 million [59].
The traditional craft industry of leather tanning
uses a series of highly alkaline and unpleasant
processes where the application of alkaline en-
zymes has brought significant process improve-
ments. There are few other examples at present of
significant uses for alkaliphiles or alkaline en-
zymes except for minor and specific applications
in the food and waste treatment industries. A
major noteworthy innovation is the complete new
industry developed by the Japanese based on
cyclodextrin glycosyhransferase from an al-
kaliphilie Bacillus sp. [2].
The production of industrial enzymes is
dominated by three companies who between them
control about 70% of the market; International
Bio-Syntheties (IBIS), The Netherlands, a joint
venture of Shell and Gist-Brocades; Novo lndustri
A / S , Denmark and Miles Laboratories, U.S.A., a
division of Bayer. There are also a number of
smaller producers supplying mainly specialist
products [60].
4.1. Detergents
The use of microbial enzymes in laundry
processes is not a new idea. For example, Biotex ®,
a ore-wash laundry detergent launched in the early
1960s contained an alkaline proteinase called AI-
calase* (Subtilisin Carlsberg) produced from
Bacillus licheniformis by N e r o lndustri. Although
enzymes in detergents soon gained popularity,
achieving a 50% market penetration by 1969 in
both Europe and the U.S. [61], subsequent con-
sumer reaction to unfavourable publicity coupled
with poor cost effectiveness and quality of perfor-
mance led to their withdrawal. However, during
the 1980s there has been a dramatic revival for
detergent enzymes due to remarkable technical
innovations and improvements. Changing patterns
of consumer demands and governmental regu-
lations fuelled partly by environmental concerns
and energy saving have contributed to their suc-
cessful reintroduction. Their commercial success
can be measured by the fact that the market share
for detergents containing enzymes is about 75% in
Europe, 55% in Japan and 45% in the U.S.A.,

where the market is forecast to reach 70% by 1995
159].
The most commonly used enzymes in detergent
formulations are proteinases and amylases. Lipase
and celhilase have also recently been introduced,
but their application is more controversial.
Proteinases have the largest market segment,
particularly sexine proteinases produced from al-
kaliphilic Bacillus spp. These are endopeptidases
with a reactive serine molecule at the active site.
Commercial products include: Alcalase ® and Es-
perase ® (Novo), and Maxatase ~ and Maxacal ~
(IBIS). Maxatase ~ is an alkaline protease active
between pH 7 and 11 with an optimum activity at
pH 9.5-10, the pH of many detergents. In its
encapsulated form it is added at 0.4-0.6~ to de-
tergents [62]. Their use is to hydrolyse proteins
and remove such obvious proteinaceous stains as
blood, egg and grass, but also less obvious stains
from body secretions, etc. Besides lifting proteina-
cenus soil, proteinases ensure that coagulated pro-
tein is not redeposited on the fabric during the
wash which gives a grey, unclean appearance to
" whites". Proteinases, including their use in deter-
gents have recently been fully reviewed [61].
Due to energy-saving consciousness and a
greater use of more delicate synthetic fabrics,
washing temperatures are continuing to decline.
The demand for proteinases has increased to com-
pensate for decreased washing performance at
lower temperatures and so has the demand for
other detergent enzymes such as amylase, lipase
and cellulase which can also improve the washing
performance.
Amylases remove starch-based stains, often
working synergistic_ally with proteinases to dis-
solve protein-starch combinations in food stains.
a-Amylases digest starch by breaking the a-l,4-
glycosidic bonds, producing soluble dextrins and
oligosaccharides. Products include Termamyl ®
(Novo) and Maxamyl * (IBIS), enzymes effective
up to 100 * C and a good performance up to pH 10
[621.
Among the more difficult stains to remove at
lower temperatures are those hydrophobic fatty
stains caused by cosmetics, body fats and oil-based
foods. Lipases degrade fats into more hydrophilic
fatty acid salts that can be removed by detergent
263
action [58]. However, their compatibility with de-
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tergant formulations is not without problems
[58,59] and their introduction is only recent [63]. If
their introduction is suCCessful and lipase-contain-
ing products prove effective, then their market
potential is as good as proteinases [59].
Cellulases have also only recently been intro-
duced as laundry enzymes, although they are more
widely accepted in Japan than elsewhere [59]. At-
tack ®, containing a celhilase from an alkaliphific
Bacillus sp. ~n4l has already achieved a 40~ market
penetration in the Tokyo area. Cdhilases exhibit
fabric softening and cohiur brightening properties
besides removing soil [58,59]. Their effect is be-
lieved to be by removing or opening up the micro-
fibrils that appear on the surface of cotton fabrics
due to wear and repeated washing, thereby restor-
ing the original appearance and smooth fibre
structure [58]. However, a weakening of the fabric
may be a problem that outweighs any washing
benefits.
For an enzyme to be an effective ~ddition to
laundry detergents it needs to be aetiv,: at alkaline
pH. Most laundry detergents have a pH in solu-
tion of between 7 and 10.5. However, the enzyme
must also be compatible with the many other
components of laundry detergents, both during
storage and in the washing process. Detergent
formulations can contain anionic and non-ionic
surfactants, eationics for fabric softening, h:each,
bleach activators, sequestering agents, anti-retie-
position agents, anti-caking agents for powders,
perfumes and optical brighteners. However, the
detergent market is very segmented due to widely
differing consumer practises which determine the
use of detergents and their formulation. For in-
stance, Americans use a short washing cycle. The
water is added at 5 0 ° C and allowed to cool
during a 10 15-rain wash at 2 0 - 3 0 ° C [61,65].
Even in Europe the boiling wash (950C) is largely
a thing of the past, but there are significant na-
tional differences. In general, European washing
conditions involve warming the water to 4 0 ° C or
6 0 ° C in a 30-60-rain cycle, with or without a cool
pro-wash [61,66]. In Japan, washing machines have
no heating element. The wash is done at ambient
tap water temperature which can be as low as 5 ° C
[67]. Detergent doses in the U.S.A. and Japan are
264

also lower than in Europe; water hardness plays a surfactants and also solvents such as ethanol (5-

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role here. Detergent components vary tOO. In the I0%), which prescott new problems for enzyme
U.S.A. cationic fabric softeners which denature stability and performance. In its liqaid form
enzymes are often added to detergents. In Europe Maxatase ® LS (IBIS) can be added to liquid de-
softeners tend to be added separately during the tergents to improve their performance !6~1.
rinsing cycle. Again, in Europe perborate and an 4.2. Leather tanning
activator such as tetraacetylethylene diamine
Traditional leather tanning relied on natural
(TAED) are used for bleaching, whilst in the
enzyme processes from bacterial growth pre~c~-d in
U.S.A. the less enzyme-compatible hypochlorite is
animal dung for dehairing and bating of hides.
preferred. All these factors affect the choice of The application of bacterial enzymes in the
enzyme for a particular application.
manufacture of leather has resulted in a quicker
it is not only consumer demands that have led and more reliable process. Conditions are highly
to changes in detergent formulae. Regulations
alkaline and this is apparently desirable for a good
governing phosphate pollution have led in some
quality finished product [2].
States to the replacement of sequestering builders
Salt cured dried hides are soaked in alkaline
such as sodium tripolyphosphate (STPP) with
liquor to swell the skins. The uptake of water and
zeolites (sodium aluminium silicates), citrate and
cleaning of the hides is improved by the addition
nitrilotriacetic acid (NTA), etc. [65]. Many of these
of detergent proteinases such as Alcalas¢ ® (Nero)
newer sequestering or ion-exchange builders
and Milezyme ® (Miles Laboratories) or special
(whose function is primarily to bind Ca 2÷ ions)
preparations such as Reverdase ® (IBIS). The en-
have either a poorer washing efficiency than STPP
zymes need to be compatible with the surfactants
which can be offset by the addition of higher
and oxidising agents which are added to assist
concentrations of enzymes, or are too expensive.
cleaning and to prevent deterioration of the hides.
Many of the first generation of commercially
The dehairing process relies on dissolving the pro-
available detergent enzymes such as Alcalase ¢
teinaeeous material binding the hair (keratin) in
(Nero) and Maxatase ~ (IBIS) perform less well in
the hair follicle without damaging the fibres (col-
the new formulations especially if the pH is higher.
lagen) of the skin, and in some cases without
Improved enzymes have been developed to meet
damaging the hair (wool) which is also a valuable
these changes, e.g. Esperase ~ and Savinase TM
product. Particularly severe conditions are ap-
(Nero), and Maxacal ~ (IBIS).
plied, especially for cow-hides, requiring the use of
Maxaeal ~, introduced in 1986 was specially
hydrated lime and sodium sulphide which causes
developed as a high-alkaline-low-temperature
unpleasant effluent problems. By adding alkaline
protease for modern detergents. It too is a serine
proteinases such as N e r o Unhairing Enzyme or
proteinase but with a high affinity for protein and
Rapidermase ® (IBIS) to a process operating at pH
an optimum around pH 11. Maxacal ® performs
8-9 the need for sulphide can be avoided or at
particularly well in low and phosphate-free formu-
least reduced. An enzymatic treatment also pro-
lations [62].
duces a higher quality product.
Another development to meet the challenge of
The bating process which is usually performed
reduced phosphate content of detergents has been
at pH 7.0-9.5 uses mixtures of proteinases. Mix-
the development of (thixotropic) liquid detergents.
tures of pancreatic and bacterial enzymes such as
Liquid detergents have several advantages over
in Batinase ® (IBIS) are used to modify the matrix
powders, such as easy dispersvl in cold water and
proteins, elastin and keratin, making the leather
convenient handling. In the U.S.A. these products
supple or rigid depending on the desired character
have already captured 38% of the detergent market
of the finished product.
[68], but thei_- acceptance in Europe has been
slower. These liquid formulations, although gener- 4.3. Other enzymes and applications
ally less alkaline (pH 7.5-9.5) than powders, usu- Deserving of special mention is cyclodextrin
ally contain higher levels of anionic and non-ionic glycnsyltransferase (CGT), which has led to the

establishment of a new industry in Japan. C G T
catalyses the degradation of starch to cyclo-
dextrins; ring compounds composed of 6-8 glyeo-
syl units linked by an a-l,4-bond. Cyelodextrins
have some remarkable properties, particularly their
ability to form inclusion complexes with organic
compounds [2]- This has led to irapoJtant applica-
tions in the food, cosmetic and pharmaceutical
industries as emulsifiers, stabilizers of volatile
flavourings, fragrances and colourants, taste mod-
ifiers and for the modification of physical proper-
lies [75]. Although eyclodextrins have been known
for many years, commercial production was not
economical until Horikoshi and Akiba [2] dis-
covered a new alkaliphilic Bacillus species produc-
ing a C G T with enhanced temperature and pH
stability.
The application of alkaline enzymes in the food
industry is not widespread. The recovery o[ val-
uable products from slaughter house waste, offal,
blood and fish waste by treatment with alkaline
proteinases, e.g. Alcalase ®, has been demonstrated
[69,70], but the technology seems to have minimal
acceptance in practice.
Large numbers of alkaliphilic Bacdlus sp. have
been isolated over the years, many due m the
systematic work of Horikoshi and co-workers.
They have probed their physiologies and molecu-
lar biology and investigated many of their en-
zymes, including amylase, xylanase, pectinase, /L
lactamase, giucanascs, iipase, pulhilanase, etc. [2],
but most of these have not been exploited com-
mercially.
4.4. Future developments
We have placed particular emphasis so far on
the detergent enzyme business because of its im-
portance in terms of volume and value. We have
also seen that it is a rapidly changing and growing
market with a great potential for innovation and
improvement. The industry is moving into the
high-technology field with a trend towards en-
zymes optimised for specific market requirements
[59]. Protein engineering is the current 'buzz-word'
and although spectacular progress has been made
[71,72] "designer enzymes" is not yet a wholly
predictable science. There is still room for the
more classical approach.
265
We must admit that up till now we have been
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remarkably conservative in our choice of environ-
ments as source material for the screening of
microorganisms for alkaline enzymes. An enrich-
ment of a soil sample at high pH almost always
yields Bacillus spp. and "normal" fertile alkaline
soils do not seem very different. Thus, it is beeo:rr
ing increasingly difficult to discover new organisms
or novel enzymes in the 'normal' environment.
Naturally occurring highly alkaline environments
such as the alkaline deserts or the soda lakes of
the East African Rift Valley are extreme environ-
ments with an enormous potential as a source of
new alkaliphiles. The Rift Valley lakes for in-
stance are extremely productive environments [5],
which contain high densities of many varieties of
microorganisms. However, as we have already said,
studies into the species diversity and the taxo-
nomic aspects of the bacteria found in these lakes
is only just beginning, but is sure to yield a wealth
of novel and extraordinary alkaliphiles. These mi-
crobes could well be the source of interesting new
enzymes and their investigation as detergent en-
zymes seems obvious.
Although there is no good a priori reason for
supposing that natural selective pressures will have
invested bacteria with good. detergent enzymes, it
is reasonable to suppose that the enzymes of ex-
treme alkaliphiles will exhibit different properties,
for how else could they function at very high pH
and high salt concentrations? Answers to such
questions on the biochemical basis of alkaliphily
will provide valuable clues for a more prediczable
basis for the protein engineering of existing en-
zymes. However, the enzymes from extreme al-
kaiiphiles may be useful detergent enzymes in
their own right. Many of the existing alkaline
serine proteinases require Ca 2+ ions, if not for
their activity then for activation and stability.
Detergents on the other hand contain sequestering
agents for Ca 2+ ions. The Rift Valley soda lakes
are low-calcium environments with concentrations
usually well below 100/tM ([5] and unpublished
results). There seems little likelihood of the
bacteria in these lakes possessing Ca2+-dependent
enzyme systems. Furthermore, the ability of such
enzymes to function under conditions of high pH
and salts may be due to an intrinsic stability,
266
which could be used to advantage in the extreme
conditions of detergent formulations - a better
stability towards auto-oxidation and autolysis for
example.
In terms of market value, leather processing
enzymes are worlh probably only 10% of the de-
tergent enzymes market. However, it is an in-
dustry that probably could profitably benefit from
high-technology enzymes. Specialised enzymes
could improve processes and provide new prod-
ucts. Alkafine lipases and amylases for defatting
and cleaning hides which, since they are salt-eared,
could be derived from haloalkaliphiles. Amylases
and specific proteinase to modify the properties of
the skins could offer advantage~. Far example,
alkaline proteases more specifically designed to
modify structural fibrous proteins instead of the
globular proteins most easily digested by present
enzyme products. Improved dehairing enzymes
could eliminate altogether the need for sulphide
and improved bating mixes could produce prod-
uets with a more predictable quality and perhaps
with new characteristics.
The catalytic properties of microbial systems
and microbial enzymes for biotransformations, and
especially for the synthesis of pure enantiomers is
receiving growing attention. There is an increasing
demand for pharmaceutical and agroehemieal
compounds to be produced in an optically pure
form and this is undonhtably one of the great
challenges for bioteehaology in the future. Indeed,
the company International Bid-Synthetics B.V.
(IBIS) was established in part to develop the
potential of this emerging new technology. Al-
though not specifically derived from alkafiphiles,
two enzymes are already marketed that function
under mildly alkaline conditions, Biofine a.M
Esterase and Biofine TM Hydratase [73].
Although microbial biotransformations are fre-
quendy promoted as having the advantage of oc-
curring under milder conditions than chemical
reactions, that is no reason why we should confine
our perspective to physiological pH and tempera-
tore. The enzymes and microbial systems of ex-
treme aikaliphiles and haloalkaliphiles may have a
spectacular role to play and could offer distinct
advantages over existing systems. The poor sohi-
bifity of some organic compounds in water can
present problems and organic solvents either pure
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or in mixed aqueous systems can reduce the activ-
ity of many enzymes. Acidic precursors will have
the advantage of greater solubility at high pH,
which may facilitate transformations by al-
kaliphiles or lead to higher rates. Other com-
pounds may have an altered charge distribution
on the molecule at high pH with perhaps surpris-
ing results for regiospecific conversion. In ad-
dition, organisms and enzymes suited to the harsh
conditions of high osmolarity and high pH may be
intrinsically more immune to the negative effects
inherent in some organic solvents. Archaebaeterial
isolates may also be worth exploring. Their chem-
istry is quite distinct and they contain some mole-
cules with the opposite ehirality to those in
Eubacteria [74].
In a certain sense extreme alkaliphiles and ex-
tremophiles in general are specialists. They have
to be able to thrive under such harsh conditions.
In the same way it is our view that the bioteehao-
logical exploitation of these organisms will be of a
specialist nature. The future probably does not lie
in application in, or modifications to existing
products and processes, but in new technologies
where the products of bioteehnotogy, be it an
enzyme or a microbial system or a reaction prod-
oct, will be suppfied as a total and discrete prod-
uct package. The applications in laundry deter-
gents are not at variance with this view, but devel-
opments will no doubt continue to surprise us.
ACKNOWLEDGEMENTS
The views expressed here are the authors' own
and do not necessarily reflect those of Gist-
Brocades or International Bid-Synthetics. We are
grateful to all our colleagues for providing infor-
mation on detergent enzymes, especially Dr. J.H.
van Ee (IBIS) and Dr. R.A. Cuperns (Gist-
Brocades).
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