Life at low water activity

19 Pages·2004·0.26 MB·English

Publishedonline12July2004
Life at low water activity
W. D. Grant
Department of Infection, Immunity and Inflammation, University of Leicester, Maurice Shock Building, University Road,
Leicester LE1 9HN, UK ([email protected])
Two major types of environment provide habitats for the most xerophilic organisms known: foods pre-
servedbysomeformofdehydrationorenhancedsugarlevels,andhypersalinesiteswherewateravailability
is limited by a high concentration of salts (usually NaCl). These environments are essentially microbial
habitats, withhigh-sugar foodsbeingdominatedby xerophilic(sometimescalledosmophilic)filamentous
fungiandyeasts,someofwhicharecapableofgrowthatawateractivity(a )of0.61,thelowesta value
w w
for growth recorded to date. By contrast, high-salt environments are almost exclusively populated by
prokaryotes,notablythehaloarchaea,capableofgrowinginsaturatedNaCl(a 0.75).Differentstrategies
w
are employed for combating the osmotic stress imposed by high levels of solutes in the environment.
Eukaryotes and most prokaryotes synthesize or accumulate organic so-called ‘compatible solutes’
(osmolytes) that have counterbalancing osmotic potential. A restricted range of bacteria and the haloar-
chaea counterbalance osmotic stress imposed by NaCl by accumulating equivalent amounts of KCl.
Haloarchaea become entrapped and survive for long periods inside halite (NaCl) crystals. They are also
foundinancientsubterraneanhalite(NaCl)deposits,leadingtospeculationaboutsurvivalovergeological
time periods.
Keywords:xerophiles; halophiles; haloarchaea; hypersalinelakes; osmoadaptation; microbiallongevity
1. INTRODUCTION a =P/P =n /n (cid:1)n ,
w 0 1 1 2
Therearetwomajortypesofenvironmentinwhichwater
wheren ismolesofsolvent(water);n ismolesofsolute;
availability can become limiting for an organism. One is 1 2
Pisvapourpressure ofsolutionandP isvapour pressure
asolutioninwhichwateravailabilityisdeterminedbythe 0
of pure water at the same temperature.
concentration of solutes in that solution, whereas in the
Water activity is simply the effective water content
other case, the availability is determined mainly by capil-
expressed as its mole fraction, which is also reflected in
laryandsurface-bindingeffects,as,forexample,inacom-
the relative humidity that is reached at equilibrium in a
plex physically heterogeneous environment such as soil.
sealed container where a hygroscopic product or solution
There is no a priori reason to suppose that an organism
has been placed.
would react differently to water stress imposed by either
It follows that pure water has a water activity of 1 and
set of conditions, but in practice, in the heterogeneous
all other solutions have values of a less than 1. The
environmentthereareothersurface-associatedeffectsthat w
advantagesofusinga includeitsreadyapplicationtosol-
complicateandcompromiseanyquantitativeanalysis.For w
utions and the ease with which it can be measured. It is
that reason, the vast majority of laboratory studies relate
less useful in complex particulate systems like soil, where
toenvironmentswherewateravailabilityisimposedbythe
adifferentterm(cid:2)isoftenused,whichaddressestheques-
presenceof solutes. A widely adopted wayof defining the
tion of capillarity. However, capillarity is negligible for
availabilityof water in a particular environment is theuse
organisms in essentially liquid environments and can be
oftheterma (wateractivity),originallydevelopedbythe
w ignoredunder these conditions. The underlying theory of
food and pharmaceutical industry, where it is used to
(cid:2)and the consideration of the capillary component for
determine shelf life and quality of product. The simple
environments such as soil is described in Griffin (1981)
water content of a material (percentage water) takes no
andGriffin &Luard (1979)and isnotconsidered further
account of water that is thermodynamically available and
in this review, which is largely concerned with homo-
has little application to the majority of situations. Water
geneous liquid environments.
activity (a ) is based on Raoult’s Law for ideal solutions
w Organisms capable of growing under conditions of low
and does not take into consideration solute interactions
wateractivityarecommonlyreferredtoundertheblanket
with components other than water; accordingly the accu-
termsxerotolerantorxerophilic (althoughseelaterinthis
racy of the calculation is greater for dilute solutions. Put
section). The suffix ‘tolerant’ indicates that the organism
simply:
is capable of growth conditions of low a , but does not
w
necessarily require low a for growth. The suffix ‘philic’
w
on the other hand indicates that the organisms actually
requirelowa conditionsforgrowth.Generally,xerophilic
One contribution of 16 to a Discussion Meeting Issue ‘The molecular w
basisoflife:islifepossiblewithoutwater?’. organisms are capable of growing at a values lower than
w
Phil.Trans.R.Soc.Lond.B(2004)359,1249–1267 1249 2004TheRoyalSociety
DOI10.1098/rstb.2004.1502
1250 W. D. Grant Life at low water activity
xerotolerant organisms, but there are exceptions to this geology of the area where they develop, for example by
general rule, particularly among the prokaryotes, where theresolutionofsaltdeposits from apreviousevaporative
thereare organismsin bothcategoriescapable ofgrowing event,orsignificantleachingofionsfromthesurrounding
in saturated salt (Vreeland 1987; Brown 1990). Two geology. The Dead Sea is an example of a hypersaline
major types of environment provide habitats for the most environment profoundly influenced by an earlier Mg2(cid:1)-
xerophilic organisms, namely foods preserved by some rich brine, somewhat depleted in Na(cid:1). Eugster & Hardie
form of dehydration or organic solute-promoted lowering (1978) have attempted to define the key geological and
of a , and saline lakes, where low a values are a conse- chemical features that influence how a hypersaline brine
w w
quence of inorganic ions. These environments are essen- develops.
tially exclusively populated by micro-organisms and Apart from total salinity and ionic composition, pH is
contain the most xerophilic organisms described to date. important in determining the composition of any
Saline soils, often adjacent to saline lakes, share micro- microbial population in any hypersaline brine. The
organisms in common with saline lakes (Ventosa et al. amount of Ca2(cid:1) (and to a lesser extent Mg2(cid:1)) is critical
1998a). in determining the final pH of a brine. The equilibrium
The stresses imposed by ions and organic solutes are between CO2(cid:3), HCO(cid:3) and CO is one of the principal
3 3 2
not necessarily the same. Many xerophilic micro- buffer systems in the aquatic environment, being, for
organisms from high-sugar foods are tolerant of low a example, oneof the buffer systems that maintains the pH
w
levels imposed by ions. Zygogaccharomyces rouxii, a xero- of seawater. The presence of Ca2(cid:1), which removes alka-
philic yeast food-spoilage organism, will grow in media line CO2(cid:3) through the precipitation of insoluble calcite
3
containing 20% (w/v) NaCl (Eriksen & McKenna 1999) (CaCO ), obviously influences this equilibrium. Brines
3
and in media supplemented with glucose or glycerol at derived from seawater have relatively high concentrations
similar a levels (although it will grow at much lower a ofCa2(cid:1)andremainaroundneutralityevenafterextensive
w w
levelsinthese media).The limitinga for food-poisoning concentration because the molarity of Ca2(cid:1) always
w
strains of Staphylococcus aureus isolated from food is the exceeds that of CO2(cid:3). Profoundly alkaline lakes develop
3
same whether the solute is salt or sugars (Scott 1957). in areas where the surrounding geology is deficient in
However, the converse is generally not true—micro- Ca2(cid:1), for example in the East African Rift Valley. Here,
organisms growing in saturated salt lakes (a 0.75) for surrounding high Na(cid:1) trachyte lavas are deficient in both
w
example, as a rule, cannot grow in media of similar a Ca2(cid:1) and Mg2(cid:1), allowing the development of lakes with
w
solely imposed by organic solutes (Kushner 1978). In pH values in excess of 11 (Grant & Tindall 1986; Grant
particular, micro-organisms inhabiting low a , high-salt et al. 1990; Jones et al. 1998). Levels of Mg2(cid:1) also influ-
w
environments have features specifically adapted to higher ence the systems by removing CO2(cid:3) as dolomite
3
levels of ions, in addition to an overarchingadaptation to (CaMg(CO ) ), and in the case of the Dead Sea, whose
3 2
low a values. Such organisms are generally described as compositionismarkedlyinfluencedbyapreviousMg-rich
w
halophilicorhalotolerantratherthanxerophilicorxerotol- evaporite,causeslightlyacidicconditionsthroughthegen-
erant. Hence, the terms xerophilic and xerotolerant are eration of Mg2(cid:1) minerals such as sepiolite, which gener-
often now restricted to describing those organisms grow- ates H(cid:1) during the precipitative process. Figure 1 shows
ingata valuesimposedbyotherthaninorganicions,and aschematic representationofthegenesisofmajorneutral
w
this usage is followedhere. There are a number ofsubcat- and alkaline hypersaline lake types. High levels of other
egories of terms for organisms that have intermediate ionsinthesurroundingtopographywillalsoinfluencethe
properties of halotolerance and halophily, not all of which final composition, and there are exceptional hypersaline
are takento meanthe same thingbydifferentauthors(see lakes dominated by Ca2(cid:1). Javor (1989) should be con-
Grant et al. 1998a). This review is concerned with organ- sultedforalistofbrinetypesandthechemicalandphysi-
isms, the majority of which are capable of growth at a cal parameters that influence their development.
w
valuesof0.80orless,whethertheybexerophilic/halophilic Surface hypersaline lakes and solar salterns are the
or xerotolerant/halotolerant. major habitats for halophilic and halotolerant micro-
organisms, but there are other, less well-studied high-salt
habitats such as hypersaline soils, salt marshes, desert
2. HYPERSALINE ENVIRONMENTS
plants,wallpaintings,seafloorbrines(suchastheAtlantis
Hypersaline waters are defined as those with total salt Deep and the Discovery Deep in the Mediterranean), oil
concentrations greater than that of seawater. Thalasso- field brines and ancient evaporite deposits, where iso-
haline waters are derived from seawater, and initially at lations of halophilic and halotolerant micro-organisms
least, have a proportional composition of ions similar to have been recorded. Accounts of these unusual environ-
that of seawater. Solar salterns, where seawater is ments and their microbial ecology are to be found in
evaporated to produce sea salt, are typical examples of Oren (2002).
thalassohaline environments. Calcite (CaCO ), gypsum Severaltermsdescribethesalttolerance/requirementof
3
(CaSO .2H O), halite (NaCl), sylvite (KCl) and finally organisms. The term halophilic is generally restricted to
4 2
carnallite(KCl.MgCl .6H O),precipitateoutsequentially those organisms that have a specific requirement for salt
2 2
as evaporation occurs. It therefore follows that the final (almostalwaysassumedtobeNaCl).Suchorganismswill
proportional composition of a hypersaline brine will be not grow in the absence of relatively high concentrations
different from that of seawater. Brines that have under- of salt, usually greater than 1.0–1.5M. Halotolerance is
gonehaliteprecipitationsaredominatedbyMg2(cid:1)andCl(cid:3) generallytaken to mean that the organismhasno specific
and are more acidic than seawater (table 1). By contrast, requirement for salt, but will continue to grow in the
athalassohaline waters are markedly influenced by the presence of high concentrations. There are examples of
Phil.Trans.R.Soc.Lond.B(2004)
Life at low water activity W. D. Grant 1251
Table1. Concentration of ions in thalassohaline and athalassohaline brines.
(Modified from Grant et al. 1998a.)
concentration (gl(cid:3)1)
seawater at seawater at Great Salt
onset of NaCl onset of KCl Lake (North Wadi Natrun
ion seawater precipitation precipitation America) Dead Sea Lake Magadi Lake Zugm
Na(cid:1) 10.8 98.4 61.4 105.0 39.7 161.0 142.0
Mg2(cid:1) 1.3 14.5 39.3 11.1 42.4 0 0
Ca2(cid:1) 0.4 0.4 0.2 0.3 17.2 0 0
K(cid:1) 0.4 4.9 12.8 6.7 7.6 2.3 2.3
Cl(cid:3) 19.4 187.0 189.0 181.0 219.0 111.8 154.6
SO2(cid:3) 2.7 19.3 51.2 27.0 0.4 16.8 22.6
4
CO2(cid:3)/HCO(cid:3) 0.3 0.1 0.1 0.7 0.2 23.4 67.2
3 3
pH 8.2 7.3 6.8 7.7 6.3 11.0 11.0
CaSO
4
gypsum
CaCO
3
calcite
salt lake pH 7–8, salt lake pH 6–7,
e.g. Great Salt Lake, Utah e.g. Dead Sea
Na+ Na+ K+ Na+
Cl– Ca2+ Mg2+ Mg2+ Cl–
high Ca2+ HCO– CO2– high Ca2+
3 3
SiO SO2– and Mg2+
2 4
Cl–
Mg Si O .nH O
2 3 8 2
sepiolite
low Ca2+
MgCO
and Mg2+ 3 CaMg(CO )
magnesite 32
dolomite
Na+ Cl–
CO2–
3
soda lake pH 10–12,
e.g. Lake Magadi, Kenya
Figure1. Schematic representation of the genesis of hypersaline brines. The centre box indicates the leaching of minerals by
CO -charged waters. Alkaline lake development is dependent on low levels of Ca2(cid:1) and Mg2(cid:1). Neutral lakes develop where
2
Ca2(cid:1) and Mg2(cid:1) levels are high. High Mg2(cid:1) lakes are more acidic due to reactions involving sepiolite precipitation. (Modified
from Grant (2004).)
organismsthatarecapableofgrowthovertherangeofsalt microbial colonization of this site (Horowitz et al. 1972)
concentrations from zero to saturation (sometimes called and the prevailing opinion is that life is unlikely to exist
haloversatile),and,indeed,othertermsfororganismsthat at this a value, which is substantially below the lowest
w
grow within a particular window of salt concentration. recorded a for growth recorded to date, that in high-
w
Kushner(1978)andVreeland(1987)haveextensivelydis- sugar food (0.61). This particular site is long overdue for
cussed the terms in use. This review is mainly concerned a re-examination using direct molecular technologies.
with organisms growing at salinities exceeding 1.0M and Primary productivity in many hypersaline lakes, mainly
doesnotattemptsuchsemanticdescriptorsfortheorgan- by halophilic and halotolerant cyanobacteria, anoxygenic
isms. phototrophic bacteria, and also eukaryotic algae of the
There is no doubt that the majority of hypersaline sites genusDunaliellamaybethesourceofthesignificantlevels
harbour significant populations of micro-organisms. oforganic compounds oftenpresent (Oren 1994;Jones et
Values of a do not generally fall much below 0.75, the al. 1998). Phototrophic productivity is probably greatest
w
limiting value obtainable at the saturation point of NaCl during periods of dilution, since most of the recorded
(5.2M).OneoftheexceptionsisDonJuanPond,asmall phototrophs, with the possible exception of Dunaliella
unfrozen Antarctic lake dominated by very large concen- spp., are unable to grow significantly at saturation point
trations of CaCl . Total dissolved salts may exceed 47% for NaCl. The organic compounds generally support sig-
2
(w/v) and the a value is recorded at 0.45 (Siegel et al. nificantpopulationsofaerobicheterotrophsthatmayform
w
1979).Therehasbeensomedisputeovertheevidencefor dense blooms that impart coloration to the brines.
Phil.Trans.R.Soc.Lond.B(2004)
1252 W. D. Grant Life at low water activity
Cyanobacteria
Acidobacteriae
Spirochaetes
Nitrospirae
Fibrobacteres
Deferribacteres
Fusobacteria
Chlamydiae
Verrucomicrobia
Planctomycetes Actinobacteria
Chlorobi
Firmicutes
Chloroflexi
Bacteroidetes root Deinococcus-Thermus
Dictyoglomi
Aquificae
Thermotogae
10%
Proteobacteria
Figure2. 16s rRNA gene sequence tree showing the major bacterial lines of descent. Lines of descent with halophilic or
halotolerant representatives are shown shaded. (Modified from Ludwig & Klenk (2001).)
Hypersalineenvironmentsarerelativelylowinoxygendue establishedgenerawithnon-halophilicrepresentatives,but
to reduced oxygen solubility (2p.p.m. in saturated NaCl, the family Haloanaerobiaceae exclusively contains halo-
compared with 7p.p.m. in seawater) and the brines also philes and the Halomonodaceae predominantly contains
harbour substantial populations of anaerobic hetero- halophiles. Table 2 lists examples of taxonomic groups
trophs. containingprokaryoterepresentativesisolatedfromhyper-
saline environments.
It is possible to make some predictions as to the roles
3. LIFE IN LOW a HYPERSALINE ENVIRONMENTS
w playedbydifferentorganismsintheutilizationandrecyc-
The upper limit of NaCl concentration for vertebrates ling of organics. Despite apparently inhospitable con-
and invertebrates is ca. 1.5M, although the brine shrimp ditions, salt and soda lakes are extremely productive
(Artemiasalina)isanexception,oftenpresentinextremely environments (particularly soda lakes, presumably
hypersaline brines but not in extremely alkaline types. because of unlimited access to CO for photosystems via
2
Eukaryotes in general are scarce above this salt concen- the HCO(cid:3)/CO2(cid:3)/CO equilibrium). Cyanobacteria and,
3 3 2
tration, with the exception of phototrophic flagellates of inneutralsaltlakes,speciesoftheeukaryoticalgaDunali-
the genus Dunaliella that frequently impart pigmentation ella are the key primary producers, although anoxygenic
to brines. Between concentrations of 1.5M and 3.0M, phototrophic bacteria of the genus Halorhodospira may be
prokaryotes become predominant, with the haloarchaea significantfromtimetotime(Grant&Tindall1986;Oren
and a few rare bacterial types such as Salinibacter ruber 1994). The primary productivity supports large numbers
forming the climax population at the point of halite pre- of aerobic heterotrophic Gram-negative bacteria. These
cipitation (Oren 1994; Anto´n et al. 2002; Grant et al. are mainly Proteobacteria, in particular members of the
2001).Afewunusualfungiandprotozoaarealsopresent, Halomonadaceae (the halomonads), probably the most
but these are probably active at lower salt concentrations importantgroupofbacterialheterotrophsinbothalkaline
(Post et al. 1983; Gunde-Cimerman et al. 2000, 2004). and neutral hypersaline environments, although other
Figure 2 indicates that the halophilic or halotolerant Proteobacteria related to pseudomonads and vibrios are
character has appeared in most of the main evolutionary alsopresent(Duckworthetal.1996;Ventosaetal.1998a;
lines of bacteria. Within the domain Archaea, halophilic Arahalet al.2002).Heterotrophic Gram-positivebacteria
prokaryotes occur in three families: the Halobacteriaceae of both the high G(cid:1)C (Firmicutes) and low G(cid:1)C
(alsoknownasthehaloarchaeaorhalobacteria)andafew (Actinobacteria) lineages are also readily isolated from
examplesofhalophilicmethanogensintheMethanospiril- hypersaline brines. Especially abundant are members of
laceae and the Methanosarcinaeae. Unlike the Halobac- the low G(cid:1)C lineage associated with the diverse Bacillus
teriaceae,wheremembersareallextremelyhalophilic,the spectrum (Ventosa et al. 1998a,b). There are also high
Methanospirillaceae and the Methanosarcinaceae have G(cid:1)Crelatives ofstreptomycetes.Isolatesfrom sodalakes
representatives that are not halophilic. Among the bac- have proven to have commercial potential in that they
teria, most halophiles described to date fall into secrete many extracellular hydrolytic enzymes, including
Phil.Trans.R.Soc.Lond.B(2004)
Life at low water activity W. D. Grant 1253
Table2. Halophilic and halotolerant prokaryotes.
groups species habitat
Bacteria
Cyanobacteria
Arthrospira platensis soda lake
Dactylococcopsis salina Dead Sea sabka
Aphanothece halophytica salt lakes
Microcoleus chthonoplastes salterns
Halospirulina tapeticola salterns (Mexico)
Proteobacteria
halomonads Halomonas elongata salterns (Spain)
Halomonas subglaciescola salt lake (Antarctica)
Halomonas halodurans river estuary
Halomonas halmophila salt lake (Dead Sea)
Halomonas eurihalina salterns (Spain)
Halomonas halophila saline soil (Spain)
Halomonas salina salterns (Spain)
Halomonas halodentrificans salterns, curing brines
Halomonas variabilis salt lake (Great Salt Lake)
Halomonas pantelleriensis alkaline saline soil (Italy)
Halomonas magadiensis soda lake (Kenya)
Halomonas desiredata sewage plant
Halomonas meridiana salt lake (Antarctica)
Halomonas campisalis soda lake (Washington)
Halomonas maura salterns (Morocco)
Halomonas alimentaria salterns, fermented sea food
Chromohalobacter marismortui salt lakes (Dead Sea)
Chromohalobacter canadiensis culture contaminant
Chromohalobacter israeliensis salt lake (Dead Sea)
Chromohalobacter salexigens salterns (Spain)
anoxygenic phototrophs Rhodothalassium salexigens brackish seawater (Oregon)
Rhodovibrio sodomensis salt lake (Dead Sea)
Rhodovibrio salinarum saltern (Portugal)
Halochromatium salexigens saltern (France)
Halochromatium glycolyticum salt lake (Egypt)
Thiohalocapsa halophila saltern (France)
Ectothiorhodospira mobilis salt lakes
Ectothiorhodospira marismortui salt lake (Dead Sea)
Ectothiorhodospira haloalkaliphila soda lakes
Halorhodospira halophila salt and soda lakes
Halorhodospira halochloris soda lake (Egypt)
Halorhodospira abdelmalekii soda lake (Egypt)
Alcalilimnicola halodurans soda lake (Tanzania)
pseudomonads/vibrios/alteromonads Pseudomonas halophila salt lake (Great Salt Lake)
Pseudomonas beijerinkii salterns?, salted beans
Marinobacter hydrocarbonoclasticus seawater
Marinobacter aquacolar oil well (Vietnam)
Salinivibrio costicola saltern? Cured meats
sulphate reducers Desulfovibrio halophilus salt lake (Egypt)
Desulfovibrio senezii saltern (California)
Desulfovibrio oxyclinae salt lake (Egypt)
Desulfohalobium retbaense salt lake (Senegal)
Desulfobacter halotolerans salt lake (Great Salt Lake)
Desulfonatronovibrio hydrogenovorans soda lake (Kenya)
Desulfonatronum lacustre soda lake (Kenya)
Desulfosalsa halophila salt lake (Great Salt Lake)
sulphur oxidizers Halothiobacillus halophilus salt lake (Australia)
Halothiobacillus kellyi hydrothermal vent (Aegean Sea)
hyphomicrobia Dichotomicrobium thermohalophilus salt lake (Egypt)
Firmicutes (low G(cid:1)C% Gram-positives)
unknown affiliation Thermohalobacter berrensis saltern (France)
haloanaerobes Haloanaerobium praevalens salt lake
Haloanaerobium alcaliphilum salt lake (Great Salt Lake)
(Continued.)
Phil.Trans.R.Soc.Lond.B(2004)
1254 W. D. Grant Life at low water activity
Table2. (Continued.)
groups species habitat
Haloanaerobium acetyethylicum oil well (Mexico)
Haloanaerobium salsuginis oil well (Oklahoma)
Haloanaerobium saccharolyticum salt lake (Crimea)
Haloanaerobium congolense oil well (Congo)
Haloanaerobium lacusrosei salt lake (Senegal)
Haloanaerobium kushneri oil well (Oklahoma)
Haloanaerobium fermentans salterns, salted fish
Halocella cellulosilytica salt lake (Crimea)
Halothermothrix orenii salt lake (Tunisia)
Natrionella acetigenica soda lake (Egypt)
Halobacteroides halobius salt lake (Dead Sea)
Halobacteroides elegans salt lake (Crimea)
Acetohalobium arabatiania salt lake (Crimea)
Haloanaerobacter chitinivorens saltern (California)
Haloanaerobacter lacunarum salt lake (Kerech)
Haloanaerobacter salinarius saltern (France)
Orenia marismortui salt lake (Dead Sea)
Orenia salinaria saltern (France)
Orenia sivashensis salt lake (Crimea)
Halonatronum saccharophilum soda lake (Kenya)
Natrionella acetigena soda lake (Kenya)
Bacillus/Clostridium
Sporhalobacter lortetii salt lake (Dead Sea)
Bacillus halophilus seawater
Bacillus haloalkaliphilus soda lake (Egypt)
Gracibacillus halotolerans soda lake (Great Salt Lake)
Gracibacillus dipsosauri salt glands of iguanas
Halobacillus halophilus salt marsh
Halobacillus literalis salt lake (Great Salt Lake)
Halobacillus thailandensis salterns, fish sauce
Salibacillus salexigens salterns
Salibacillus marismortui salt lake (Dead Sea)
Oceanobacillus iheyensis deep sea ridge
Tindallia magadiensis soda lake (Kenya)
Clostridium halophilus salt lakes
Desulfotomaculum halophilus oil field brine (France) sulphate
reducer
cocci Salinicoccus roseus salterns (Spain)
Sailinicoccus hispanicus salterns (Spain)
Marinococcus halophilus salterns
Marinococcus albus salterns
Tetragenococcus halophilus salterns/salted food
Tetragenococcus muraticus salterns? fermented squid
Actinobacteria (high %G(cid:1)C Gram-positives)
Nesterenkonia halobia salterns
Actinopolyspora halophila culture contaminant
Actinopolyspora mortivallis saline soil (Death Valley)
Actinopolyspora iraqiensis saline soil (Iraq)
Nocardiopsis lucentensis saline soil (Spain)
Nocardiopsis halophila saline soil
Nocardiopsis kunsanensis saltern (Korea)
Bacteroidetes
Flavobacterium salegens salt lake (Antarctica)
Salinibacter ruber salterns (Spain)
Archaea
haloarchaea Halobacterium salinarum salterns/salted hides
Haloarcula vallismortis saline ponds (Death Valley)
Haloarcula marismortui salt lake (Dead Sea)
Haloarcula hispanica salterns (Spain)
Haloarcula japonica salterns (Japan)
Haloarcula argentinensis salt flats (Argentina)
(Continued.)
Phil.Trans.R.Soc.Lond.B(2004)
Life at low water activity W. D. Grant 1255
Table2. (Continued.)
groups species habitat
Haloarcula mukohataei salt flats (Argentina)
Haloarcula quadrata sabka (Egypt)
Halobaculum gomorrense salt lake (Dead Sea)
Halococcus morrhuae salterns? salted cod
Halococcus saccharolyticus salterns (Spain)
Halococcus salifodinae salt mine (Austria)
Haloferax volcanii salt lake (Dead Sea)
Haloferax gibbonsii saltern (Spain)
Haloferax dentrificans saltern (California)
Haloferax mediterranei salt lake (Spain)
Halogeometricum borinquense saltern (Puerto Rico)
Halorhabdus utahensis salt lake (Great Salt Lake)
Halorubrum saccharovorum saltern (California)
Halorubrum sodomense salt lake (Dead Sea)
Halorubrum lacusprofundi salt lake (Antarctica)
Halorubrum coriense saltern (Australia)
Halorubrum distributans saline soil (USSR)
Halorubrum vacuolatum soda lake (Kenya)
Halorubrum trapanicum saltern (Sicily)
Halorubrum tebequense salt lake (Chile)
Haloterrigina turkmenica alkaline soil (USSR)
Haloterrigina thermotolerans saltern (Puerto Rico)
Natrialba asiatica beach sand (Japan)
Natrialba taiwanensis saltern (Taiwan)
Natrialba magadii soda lake (Kenya)
Natrialba hulunbeirensis soda lake (China)
Natrialba chahannoensis soda lake (China)
Natrinema pellirubrum saltern? salted hide
Natrinema pallidum saltern? salted cod
Natrinema versiforme salt lake? (China)
Natronobacterium gregoryi soda lake (Kenya)
Natronobacterum nitratireducens soda lake (China)
Natronococcus occultus soda lake (Kenya)
Natronococcus amylolyticus soda lake (Kenya)
Natronococcus pharaonis soda lake (Kenya)
Natronorubrum bangense soda lake (Tibet)
Natronorubrum tibetense soda lake (Tibet)
methanogens Methanocalculus halotolerans oil well (France)
Methanohalobrium evestigatum saline lagoon (Crimea)
Methanohalophilus mahii salt lake (Great Salt Lake)
Methanohalophilus halophilus salt lake (Australia)
Methanohalophilus portucalensis saltern (Portugal)
Methanosalsum zhilinae soda lake (Egypt)
Methanococcus doii saltern (California)
proteinases, cellulases and lipases (Rees et al. 2003). Sulphidogenesis is usually pronounced in hypersaline
Genencor BV (Leiden) currently markets two different sediments. Isolation of halophilic sulphate-reducing
soda lake cellulases derived from Gram-positive isolates bacteriahasprovendifferent,butthere arenowseveralof
for use in laundry and textile processes. these in cultures from both neutral and alkaline
Hydrolysis products of complex polymers are also sub- hypersaline environments including Desulfovibrio,
strates for anaerobes, especially members of the Halo- DesulfonatronovibrioandDesulfobacterspecies(Caumetteet
anaerobiales and organisms related to other clostridial al. 1991; Zhilina et al. 1997; Tsu et al. 1998). Oxidation
groupsinthelowG(cid:1)CdivisionoftheGram-positivebac- of sulphide is brought about by anoxygenic phototrophic
teria.Thesebacteriafermentorganiccompoundstoacetic bacteria such as Halorhodospira spp. and under aerobic
acid, hydrogen and CO (Grant et al. 1998a; Zavarzin et conditions inalkaline hypersaline lakesby Thioalkalivibrio
2
al. 1999), which in turn may be used by methanogens, spp. (Sorokin et al. 2001). Halophilic thiobacilli are also
although most of the methanogens isolated to date from found in neutral salt lakes (Kelly & Wood 2000).
hypersaline environmentsare methylotrophic, using com- Ammonia oxidation is known to take place in soda lakes
pounds such as methanol and methylamine (Oren 1999). (Khmelenina et al. 2000), but nitrifying bacteria have not
Such C1 compounds are likely to be abundant in hyper- yet been recorded in any neutral lakes.
saline brines, probably derived from the anaerobic The climax population in sodium-dominated hyper-
decomposition of cyanobacterial mats. saline lakes at the point of halite (NaCl) precipitation
Phil.Trans.R.Soc.Lond.B(2004)
1256 W. D. Grant Life at low water activity
almostalwayscompriseshaloarchaea,withonlyafewbac- Table3. Approximate water activity (a ) values of selected
w
terial types such as S. ruber able to compete. These satu- foods.
rated brines provide among the most extreme a values (Adapted from Brewer (1999).)
w
possible via inorganic solutes. Soda lakes impose the
additional stress of very alkaline pH values, up to pH 12 aw value foods
in some cases. Haloarchaea now comprise 15 genera
1.00–0.95 fresh meat, fresh and canned fruit and
(table 2) on the basis of phylogenetic analysis, although
vegetables, sausages, eggs, margarine,
phenotypically they are all rather similar. Dense blooms
butter, low-salt bacon
of these organisms colour neutral and alkaline saturated
0.95–0.90 processed cheese, bakery goods, raw ham,
hypersalinelakesbrightred.Theseorganismsarethemost
dry sausage, high-salt bacon, orange juice
halophilic known, most requiring at least 2M NaCl for concentrate
growth and many capable of growing in saturated NaCl 0.90–0.80 hard cheese, sweetened condensed milk,
(5.2M). As might be expected, the isolates from alkaline jams, margarine, cured ham, white bread
soda lakes have an additional requirement for high pH in 0.80–0.70 molasses, maple syrup, heavily salted fish
growth media, usually growing between pH 8.5 and 11.0 0.70–0.60 Parmesan cheese, dried fruit, corn syrup,
with an optimum at pH 9.5–10.0, whereas those halo- rolled oats, jam
archaea from neutral lakes generally have pH optima for 0.60–0.50 chocolate, confectionary, honey
0.40 dried egg, cocoa
growth between pH 6 and pH 8. Alkaliphilic haloarchaea
0.30 dried potato flakes, potato crisps, crackers,
are currently classified in six genera (table 2); four of
cake mix
these, Natronococcus, Natronobacterium, Natronorubrum
0.20 dried milk, dried vegetables
andNatronomonas,harbouronlyalkaliphiles.Haloarchaea
from neutral sites comprise the remaining 11 genera, two
of which contain both alkaliphilic and neutrophilic types.
Itisnotusuallyclearwhetherbloomsofthesehaloarchaea becomeentrappedwithinthecrystals,leavingbehindaso-
comprise predominantly one species or a mixture of sev- calledbitternbrine dominatedby MgCl and KCl,which
2
eral species. Often a majority of isolates from neutral does not support significant growth of haloarchaea,
brines are Halorubrum or Haloarcula spp. (W. D. Grant, although it is not actually toxic (Norton & Grant 1988).
unpublishedresults),butananalysisoflipidsinDeadSea The failure to recover haloarchaea from these bittern
biomasssuggestedmainlyHaloferaxandHalobaculumspp. brines is because these have been physically removed by
(Oren&Gurevich1993).Earlyworkreliedonphenotypic entrapment within halite. Crude solar salt contains many
characterization, and most rod-shaped isolates were viable haloarchaea, typically 106 viable cells per gram.
assignedtothegenusHalobacterium.However,Halobacter- Since crude solar salt is generally used in the salting of
iumsalinarum, the solerepresentative ofthis genus, issel- hides and fish, this is the explanation for the recovery of
dom isolated from hypersaline brines and is typically haloarchaea from these products, notably Halobacterium
isolated from salted hides and salted fish, as a conse- salinarum.
quence of its proteolytic capacity. Alkaline brines have
yielded a considerable number of isolates, but it is not
4. LIFE IN LOW a FOODS
clear as yet, which types, if any, are dominant. w
Direct molecular analysis of both alkaline and neutral Low a foods fall broadly into two types: cured foods
w
hypersalinebrinesby16SrDNAamplificationofenviron- whosewateractivityisloweredbythepresenceoraddition
mental DNA, preparation of gene libraries, followed by of a solute, either salt or sugar, and those foods that are
sequence determinations of individual 16S rRNA genes, dehydrated by the removal of water by freeze-drying or
hasrevealednovellineagesthathaveyettobebroughtinto simple evaporation. Freezing food owes part of its effec-
culture (Grant et al. 1999; Benlloch et al. 2001), notably tiveness to the removal of water. Clearly, other factors
enabling the phylogenetic identification of the square flat suchastemperature,pH,oxygenandthepresenceofanti-
cells first described by Walsby (1980) that are frequently microbial inhibitors also markedly affect any microbial
observed in neutral hypersaline brines from a variety of population (Houtsma et al. 1996). Table 3 lists the a
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geographical locations (Anto´n et al. 1999). There have values for particular types of food.
alsobeenreportsoftheretrievalofhaloarchaealsequences Preventing pathogen growth and retarding spoilage are
from environments that are not particularly saline, sug- crucialinthepreservationoffoods.Mostfreshfoodshave
gestingthatlesshalophilichaloarchaeamayexist(Munson a valuesof0.95–0.99,allowingthegrowthofmanytypes
w
et al. 1997). of micro-organism. The minimum a for most bacteria is
w
Haloarchaealbloomsinsolarsalternsareknowntopro- ca.0.90(table4)andfoodswitha valuesbelowthispro-
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mote crystallization of halite. It is possible that the cells vide environments largely for xerophilic and xerotolerant
may serve as templates in the nucleation of halite crystals fungi and a few highly resistant prokaryotes (Brewer
and their subsequent development (Lope´z-Corte´s & 1999). The vast majority of human pathogens are sup-
Ochoa1998).Thereisnodoubtthatsaltyieldsaredimin- pressed by a values below 0.90 (Houtsma et al. 1996).
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ished in the absence of haloarchaeal blooms, and labora- OneexceptionisStaphylococcusaureus,whichisextremely
tory experiments support this view (Javor 2002). xerotolerant and will survive in salted foods at a values
w
Haloarchaeal blooms, by virtue of their red carotenoid ofca.0.82(15% w/vNaCl)(Scott1957; Kushner1978).
pigments, also increase light absorption of the brines and Curing foods in baths of brine extracts nutrients that
promote evaporation by increasing the temperature. promote the growth of halophilic and halotolerant micro-
Observationsofhalitecrystallizationshowthathalorchaea organisms. Often, specific concentrations of salt are
Phil.Trans.R.Soc.Lond.B(2004)
Life at low water activity W. D. Grant 1257
Table4. Minimum inhibitory a values for growth of micro-organisms in food.
w
(Adapted from Brewer (1999).)
a value prokaryotes yeasts moulds
w
0.97–0.95 Clostridium spp.
Pseudomonas spp.
Escherichia spp.
Bacillus spp.
Pediococcus spp.
Citrobacter spp.
Vibrio spp.
Lactobacillus spp.
0.95–0.90 Streptococcus spp. Rhodotorula spp. Rhizopus spp.
Corynebacterium spp. Pichia spp. Mucor spp.
Micrococcus spp. Candida spp.
Trichosporon spp.
0.90–0.85 Staphylococcus spp. Saccharomyces spp. Cladosporium spp.
Hansenula spp.
Torulopsis spp.
0.85–0.80 Zygosaccharomyces bailii Aspergillus patulum
0.80–0.75 haloarchaea Aspergillus glaucus
Aspergillus conicus
Aspergillus flavus
(cid:4)0.70 Zygosaccharomyces rouxii Xeromyces bisporus
necessarytofavourasuccessionoforganismssuchaslac- 1985). Growth of Z. rouxii has been recorded at a 0.62
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tic acid bacteria during fermentation, for example in andtheorganismalso,unusually,iscapableofgrowingin
picklesandsausages(Brewer1999).Atveryhighsaltcon- relatively high NaCl concentrations (20% (w/v); a 0.85;
w
centrations (a values below 0.8) the only prokaryotes to Tokuoka(1993)), sharing with X.bisporus the capacityto
w
flourisharethehaloarchaea,originallyintroducedintothe produce ascospores at a values below 0.7.
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foodviasolarsalt.Thesemaycausethespoilageofheavily
salted proteinaceous products, being responsible for the
‘pink’insaltedfishand‘redheat’ofsaltedhides(Grant& 5. OSMOADAPTATION
Larsen1989).Here,oneparticularspecies,theproteolytic
Micro-organismsexposedtoalowa environmentmust
H. salinarum seems to be dominant. w
possess mechanisms to avoid water loss by osmosis. At
The minimum a that can be achieved by the addition
w least some level of turgor pressure has to be maintained
ofNaClis0.75(saturationpointforNaCl).Valuesbelow
toallowcellsurvivalandgrowth.Therearetwobasicstra-
this infood can occuronlyinthepresenceoforganic sol-
tegies:
utes such as glucose and fructose, and sometimes there
may be additional significant surface effects in hetero-
(i) a minority of halophiles use counterbalancing levels
geneous foods through drying. Foods, which include
of inorganic ions (usually KCl) to achieve osmotic
honey,fruitsyrups,jams,marmaladesanddriedfruits,are
stability;
dominated by xerophilic fungi and yeasts. The vast
(ii) the majority of halophiles (and all the xerophiles)
majority of fungi and yeasts are inhibited at a values
w produce or accumulate low-molecular-mass organic
between 0.8 and 0.75, but the spoilage mould Xeromyces
compounds that have osmotic potential.
bisporus is exceptional in being able to grow at a lower
wateractivity(a 0.61)thananyotherorganismdescribed
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to dateand can thus cause spoilage offoods thatare nor- These osmolytes, also known as compatible solutes
mally considered safe from microbial attack (Hocking & because of the need for them to be compatible with cell
Pitt 1999; table 4). The organism will not grow at a machinery(Brown1978,1990),mustalsoprotectagainst
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valuesgreaterthan0.97,isthusanobligatexerophile,and inactivation, inhibition and denaturation ofboth enzymes
is also capable of completing a sexual life cycle, forming and macromolecular structures under conditions of low
ascospores, at a values below 0.7. Originally isolated water activity. Compatible solutes are polar, normally
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from spoiled liquorice, X. bisporus also causes problems uncharged or zwitterionic compounds under the con-
with dried fruit, tobacco and honey, although its natural ditions experienced inside cells. Compatible solutes
habitatisasyetunknown(Hocking&Pitt1999).Zygosac- belongtoseveralclassesofcompoundsandtherearesome
charomyces rouxii is one of three Zygosaccharomyces spp. commonstructuralmotifs,particularlyamongaminoacid
that are troublesome spoilage organisms at very low a derivatives.Figure3showsthemostimportantcompatible
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values, particularly under acidic conditions in products solutes of micro-organisms. They include the following:
such as fruit concentrates and brined vegetables
(Eriksen & McKenna 1999). Under extreme conditions, (i) polyols such as glycerol, arabitol, mannitol, sugars
internalCO pressureduetosugarfermentationcancause or sugar derivatives such as trehalose, sucrose, sul-
2
explosionsofglassbottlestooccur(Thomas&Davenport photrehalose and glucosylglycerol;
Phil.Trans.R.Soc.Lond.B(2004)
1258 W. D. Grant Life at low water activity
CH3 CH COO– O NH+3 O
2
CH3 N+ CH3 NH2 C CH2 CH CH2 COO– CH3 C NH CH2 CH2 CH2 CNH+3
H COO–
glycine betaine β-glutamine Nδ-acetylornithine
O
OH
NH NH
C CH CH CH
2 2 2
CH + COO– CH + COO– CH3 NH CH2 CH COO–
3 NH 3 NH
NH+
3
ectoine β-hydroxyectoine Nε-acetyl-β-lysine
CH2OH CH2OH CH2OH
OH O OH O OH O
OH OH OH
O
OH OH
OH
O CH OH O SO3H O
2
OH
O CH OH O CH2OH
2
CH OH O OH OH OH OH
2
OH OH
sucrose trehalose 2-sulphotrehalose
CH OH
2 CH
OH 3
O NH
CH S CH 2
OH 3 + 2 COO– O C
CH OH CH
OH 2 2
CH
2
O C H dimethyl sulphonio- O
CH
propionate 2
CH OH C NH C CH
2 3
glucosylglycerol NH O C
2
C CH NH
O 2
CH C
2
O H
NH + C O
3
C CH CH C
2 2 COO– NH2
CH3 NH CH2 CH2 H Nα-acetylglutaminylglutamine amide
glucosylglycerol
NH
2
C O
H
NH
2
H C OH C CH NH CH CH COO–
O CH 2 C 2 2
H C OH 2
H
H C OH C O CH2 + C
N
NH H H
H 2 2
glycerol Nα-carbamoylglutamine amide proline
Figure3. Structure of compatible solutes. (Modified from Grant et al. (1998a) and Oren (2002).)
Phil.Trans.R.Soc.Lond.B(2004)