Psychrophilic (cold-adapted) microorganisms make a major contribution to Earth’s biomass and perform critical roles in global biogeochemical cycles. The vast extent and environmental diversity of Earth’s cold biosphere has selected for equally diverse microbial assemblages that can include archaea, bacteria, eucarya, and viruses. Underpinning the important ecological roles of psychrophiles are exquisite mechanisms of physiological adaptation.

1. Journal or Scicnti ric & Industri,iI RcscOirch
vol. St). Fchruary :W()(). pp X7- IOI
Biotechnological Application of Psychrophiles and
Their Habitat to Low-Temperature
K V Ramana*, Lokendra Singh and R K Dhaked
Bi otcchnology Di vision. Defcncc Rcscarch ,lIld Dcvclopmcnt
EstOlhli shmcnl. Jhansi Road. Gwalior 474 ()()2. lndia
Rcccived : 16 M ;lrch 1l)l)'J: accepted: 19 November I99()
MiCl"oorg; lIli~m s li ving in extrcme environments ;lre di vided into ri ve categorics: thcrmophilcs. psychrophiles. alb liphiles.
ilcitiophi Ics ;lIld halophiles. Environments that are considcrcd to heextremc are colonized by th e ~e special microorganisms which
OIre ;Idapted to these ecologic;iI niches. Thc applic,lIion or psychrophilic microorgani sms in industrial processes opens up a neV
cra ill hiotechnology. Due to its unique hiochemieal reatures. it can be ex ploited ror use in biotechnologic,iI industri es busy in
m<lnur;lcturing rood. enzy mes. and value added pharmaceuticals products. Recent devclopments show that psychrophiles arc
good source or novel catalysts or industrial interest. Some or the enzymes have been isol<lted and the genes arc sueeessrully
cloned and ex pressed in target hosts. Biopolymer degrad ing enzy mes like amylase. pullulanases. xylanases. ;lIld proteases play
;In import,lnt role in rood. detergent s. and pulp industries. Cell membranes or psyehrophiles contain surractanls hearing unique
s(;lhil ity at low temperatures ;lIld th;1I Gin he used in pharmaceutical rormulation. The cold adaptation process or psychrophiles
encompasses wide Illodirications in structun.: ami molecular architecture. physiology. and hiochemist ry or these organisms.
These arc desnihed. in det;lil. here.
Introduction
At low temperature. growth and development of
microorganisms is drasticall y reduced due to the low
solu bility of organic and inorganic nutrients, decreased
ion/solute transport , reduced diffusion, and osmotic ef-
fects on plasma membrane and cd I wall I . Psychrophi Iic
bacteria are most widely distributed than other types of
cxtremoph iles for the si III pic reason that water covers
approx. 70 per cent of the cart h's surface and a large part
of it constitutes the marine environment. About 90 per
cent of the mari ne habitat ex ist at < SoC beyond the
thermocline zone of oceans2
. Apart fromthe marine
habitat , polar regions measure nearl y 14 per cent or the
eart h's surface, representing an extreme low-tempera-
ture habi tats in the world. The growth characteristics of
psychruphiles and psychrotTophs make them suitable to
grow in variolls low-temperature habitats i.e., in lake
sediments, ;tS gut nora or aquat ic animals, and in the
form or periphytic Illicronora, and on rocks at low
ll1uisture conditions. Psychrophilic bacteri a arc highly
thermnsen .~ iti ve and ex p()sure to moderate room tem-
perat ure could he detrimental to their acti vit y. Most or
the psychrophiIic hacteria thus far isolated are gram-
negati'e and reports on the occ urrence of gram positi ve
':'Aut hor ror cmrespondencc
bacteria are relati vely few. To exemplify their diversity.
various bacteria belonging to different genera have been
isolated in the recent past.
Extrer1'lophiles and their enzymes have attracted
much attention because of their wide range of biotech-
nological applications and also to understand their bio-
che mi cal mec hani sms of adaptat ion to ex tremes
temperature, pH and salinity. Further, they are being
utili zed in various bioprocesses to be carried out at low-
temperature, in addition to their role in natural decolll-
position of organic matter and nutrient recycling at low
temperature habitats. Particularly, studies on psychro-
philic and psychrotrophic microorganisms are relati vely
sparse in comparison to thennophiles and hypenherl11o-
philes. According to the definiti on of Morita~, psychro-
philes show an optimum growth temperature of < ISoC
with upper growth temperature of 20°C, whereas psy-
chrotrophs can grow at (j°C although their optimulTl
temperature is around 20-2S°C. Psychrotrophs wen:
further classified as stenopsychrotrophs and eurypsy-
chrotrophs based on theiI' abi Iity to grow at 4()OC (rer.4)
Stenopsychrotrophs arc able to grow sub-optimall y at
4()OC in contrast to eurypsychrotrophs. Conspicuollsly
the characteristic growth rate of these microorganisms
is not much affected at low temperature.

2. xx J SCIIND RES VOL S9 FEBRUARY 2000
The ability of psychrophilic and psychrotrophic bac-
teria to grow at low-temperatures is due to the stabi lity
of cell constituents such as enzy mes, fatty acid compo-
sition of lipids in cell membranes, and modifications in
nucleic acid constituents and control over the gene ex-
pression through the mediation of transcription and
translation with the production of cold-shock proteins.
In the biosphere, both prokaryotes and eukaryotes are
known to ex ist as psychrophiles. In addition to polar
regions fresh water lakes. rivers, marine sediments and
deep sea water are the best source of these bacteria. In
addition to the isolation of bacteria, several types of
yeasts. fungi and algae have also been reported. These
microorganisms were found to be responsible for min-
eralization and nutrient recycling at as low as _l aC in
maritime Antarctica.
The presence of a wide variety of hydrolases namely
amylase, protease, cellu lase, nuclease, lipase, ~- galac-
tosidase, and phosphatase have been reported from Ant-
arctic bacteri a,,·7 This rev iew discusses the role and
imporlance of psychrophilic microorganisms in the
biodegradation of organic pollutants. In add ition, vari-
ous basic aspects of cold adaptation and biotechnologi-
cal potential of metabolites and hydrolytic enzymes
have also been reviewed.
Phylogeny of Extremophiles
The phylogeny of extremophi les is based on their 16S
rR A sequence and the uni versal tree has been divided
into bacteria, archae and eukarya. The archae consist of
two major kingdoms, the crenarchaeota-thermopro-
teales branch and the euryarchaeota. The latter kingdom
consists of extremophiles and methanogens
N
The start-
ing lineage of bacteria is represented by the orders
thermotogales and aquificiales which again represent
hyperthermophi les'} Signi fi cant Iy, most of the psychro-
philic bacteria have been assigned to bacteria. Excep-
tionally, a few representative species of crenarchaeota
and euryarchaeota have been reported with the help of
polymerase chain reaction and genecloning experi-
ments10. Some of the recent Iy isolated antarctic bacteri al
isolates belongs to Cylop/lOga-F/a vobaclerilll11- Bac-
leroide. and they have been classified as Polaribacler
fral7~I1/(/l/ii strain 3() I and Po/arihacler.fi/amenllls strai n
2 15 based on their 16S rRNA sequence analysis. Mosl
of the bacterial strains isolated from Antarctica have
been classified phylogenetically based on the sequence
homology of their 16S ri bosomal DNA or RNA indicat-
ing the presence of many new and previously unreported
orga ni sms that inc lude P/anoc()CCUS IIIcmeekinii,
Arthrobacler, Brachybacteriul1l and PsychroflexlIs IOr-
. 11 .12
qUiS gen, nov., sp.nov.
Habitat in Relation to Structure and Molecular
Architecture
Ecological Sign!/icance and Distribution
It has widely been noticed that in cold habitats,
metabolic role of these microorganisms is essential in
the biodegradation of organic matter and for recycling
various carbonaceous and nitrogenous nutrients in large
quantitiesl ~. I~. Most of the organic ni trogen released in
natural habitats under extreme low-temperature is pre-
sent in the form of hi gh molecular weight proteins,
nucleic acids along with uric acid and urea which cannot
be utilized directly by the nati ve bacterial populations.
Proteins can only be utilized by bacteria after extracel-
lular hydrolysis by proteases.Several p. ychmphiles that
produce substantial amounts of extracellular enzy mes
such as proteases, lipases, amylase, nucleases, phos-
phatase, cellulase, and ~-galactos idases have heen im-
pi icated in biodegradat ion. 1n low-temperature habitats.
comparati vely large amount or enzyme is synthesized
and secreted to compel.;ate the ciecrea<;ing enzy me ac-
ti vity. The phenomenon was noticed in the case of
hydrolytic enzy mes such as phosphatase, nuclease,
amylase, and lipase l'i. 17. Nutrient rich media with suit-
able inducer substrate molecules are effecti ve to stimu-
late the synthesis of higher quantities of enzy mes.
Various enzymes produced by these bacteria are listed
in Table I along with their optimum temperature and pH.
Also the quantity if enzyme is known to affect the rate
of biodegradation. Usually, hydrolysis by extracellular
enzymes is often a rate limiting step for bacterial utili-
zation of organic nitrogenous and carbon substrates lx.I'}.
Some Unique Features 0/Bacteria (lnd their EII~vll1es
Psychrotrophic bacterial isolate of Vihrio sp strai n
5709 has an optimum growth at 20°C. The bacteria was
isolated from a deep sea sedi ment and the protease from
this strain was optimally acti ve at 40°C. Retention of
high enzyme activity at low temperatures is a charac-
teristic feature of psychrophiles. For example. 16, 3"2,
and 45 per cent of optimal activity was observed at ()O,
100, and 20°C respecti vely.
Alkaline phosphatase (EC 3. 1.3. 1) is one of the hy-
drolytic enzymes which is required for phosphorylation
. I I b' I 10 EIn mo ecu ar 10 ogy- . nzymes such as nitrate reduc-
tase and arginino succinate I ya~e from psychrophil ic
green algae belonging to the genus Ch/oml1IOIWS have

3. RAMA l A l'11I1. : BIOTECH OLOGICAL APPLICATIO OF PSYCHROPHILES X9
Tahle I - Temrerature and pH ort ima or r sychrurhilic enzy me~ Tahle I - Temrerature and pH ortima or rsychrophilic enzyme ~
...conld
Microorganism
PS(' lldO/llfIIWS
{/{TlIg ill{).1"lI
Sl'rmlill
/iqlll'fi u'il'lls
i!lIelllllls sp
Enzyme
Alkaline rrot ea ~e
Lipa ~c
Protease
Prot ea~e
Lipa~c
IJSl'lIdOIlWIWS sr Protea~e
Lipa~e
VI'I'lIdOIlI{)lIl1S ~r Prot e a~c
X{IIIIIUJlIIOIIOS
1I1l1110l,hilu
Prot ea ~c
COlldidu hlllllico/u Prot ea~c
/Jucilllls co"gll- Proteasc
IIIIIS
I'S{,IIII{)II/OIIIIS ~p Li p : ~C
PSI'//{"J/IIIJ/WS
lolllsii
Ar{)III{)II(/S
hwlrol",illl
Alle'lmlio/llIS
hll"'I'/IIII('lis
MieT()C{)CCIIS sp
IVlicr{)('(J('CIIS sp
Micr{)('()(n IS ~ p
tl rthrohllcll'r ~ p
Li PiN:
Am y l : ~e
Prot ea~e
a- Amyla~c
Amylase
o.- Am y la ~c
Amylase :lI1d
pullulanasc
Alkalinc
phosphata~c
Alkalinc
ph(l~pha t asc
Optimum
temr/rH
SSOC
pH X.O
]SOC
pH 70
4S0C
pH 7.0-11.0
pH X.O-9.0
2S0C
pH Y.O- I0.0
R er~
2X
X4
X4
X4
107
lOX
109
11 0
III
11 2
II ]
11 4
li S
117
20
Microorganism
Vibrio II/OrillllS
P~ychror h ile
F/0'()/){Iell'ri11111
Vi /Jrio sp
Psychrophile
RllOdococcliS sp
Enzyme
Triose- ph o~ ph ate
i somera~e
~- I aclama~e
~- mannana~c
Aspartate
transcarhamoyl
tran s rera~e (ATCa~e)
ATCasc
Aliphatic aldehyde
dehydrogenase
PSl'lIdOIlIOIWS sp 9-hexadccenoic acid
ci~- t rans i ~o mc ra ~e
Ct'lwu'hllt'11I11
.IT lll bioSIIIII
N ilrOSOI 'ibrio ~p
Vibrio sr
PSl'lldOIIlOllllS
.f/1/{)r{,S(·('IIS
Vihrio ~ p
D A pol Y lll era~c
Ru BisCo
Hydroxylamine
oxida~e
ATPase FOFI-tyre
Rihonu c l ca~c
]-isopropyl malate
dehydrogcn:Sc
Optimum
temr/pH
SO°C
ph S .] and
X.O
A II;mIlWlIlIS sl'
K 14-2
Tryptoph:Il ~y ntha~e 10°C
Arlhrohllc/{'r ~p ~-galact(ls i d;~e
Refs
II X
IIY
27
94
42
120
121
10
122
22
7
123
been investigated and their properties compared with
enzymes obtained from a mesophilic Chlamfdn/llOn{fs
reinhardtii. The e n zy me.~ were found to be cold acti ve
and thermolabile.The enzy mc arginino succinate lyase
(ASL) retained 2S per cent of its ori ginal activit y at 4°C
whereas mesophilic enzy me was completely inactivated
" 1
at the same temperature- . The study also revealed that
Ch/nr()fl1()fws nitrate reductase and ASL, displayed dif-
ferent thermal properties.The former enzy me was found
to be more thermolabile with 50 per cent of its acti vi ty
loss after incubation at 4()OC for jO minH
. More often,
it was found that enzymes from an organism can di splay
cOl1td diffcrent temperature and pH optima along with varying
stability.

4. l)() J SCIIND RES VOL 59 FEBRUARY 20()()
The chitinase product ion is mainly attributed to the
biodegradation of large quantities of chitin produced by
in vertebrates that inhabit the water column and sedi-
ments of oceans and estuaries. The exocellular chiti-
nases hydrolyze chitin into microparticles. oligomers
and other small molecular weight sugars which can be
further metaboli zed by bacteria as carbon and energy
source2
,.2.J. Apart from chitin the biomass resulting from
sea weeds and animal activity is continuously decom-
posed by microorganisms mainly by psychrophilic bac-
teria.
In the marine ecosystem several of chitinoclastic
bacteria have been reported in water and sediments of
the antarctic maritime peninsula. In our earlier in vesti -
gations. we have categori zed antarctic psychrotrophic
bacteria isolated from deC<lying blue green al gal mass as
acidotolerants, alkalitoierants. and thermotolerants. The
bacterial isolates were able to grow at higher salt con-
centrations (up to 10.0 per cent) in the growth medium
signifies that these isolates are hal otolerant in their
nature'7
Antarctica psychrophilic bacteria display remarkable
act ivity at O°e. Bacteria belonging to the genus Psychro-
/JoCl('/' sp are predominantly found in antarctic ornitho-
ge nic soils" .
Many enzy mes from psychrophiles display substan-
tial acti viry at very low-temperatures. For example an
acid phosphatase with maximal activity at pH 6.0 and
~ O°C has displayed 27 and 28 per cent of its maximal
acti vity at 0 and 5°C, respecti vely25 Several other en-
zy mes have also been noticed to show high catalytic
activity at low and intermediate temperatures with rapid
inact ivation at or above 40°e. Subtilisin from psychro-
ph iI ic antarct ic bacteria has been well characterized in
comparison to otherenzymes2(,. Hydrolytic enzy mes arc
essential for processing the huge quantities of organic
mattcr. mainly in marine habita!. Bacterial consortia
with different but complementary ex tracellu lar hydro-
lytic enzyme profil es act on organic particles to rapidly
solubilize major macromolecular constituents. Hydro-
lytic enzy me activities such as protease, phosphatase,
glucosidase and other enzyme activities are responsible
for enzymatic fractionation of carbon, nitrogen, and
phosphorous containing organic wastes and the produc-
tion or these enzymes is mainly attributed to attached
I . I I ' '7lLlctena popu atlons- .
Bacteria present in marine snow have cell specific
ectoenzy me activities several-fold or magnitude higher
than bacteria present in the surrounding water. The
phenomenon is indispensable for optimum growth even
in the presence of low-quantities of nutrients in thei r
I b· ?X-10 S I I I
la Itat- - . evera enzy mes are a so t;nown to he pro-
duced extracellularly by psychrophilic microorganisms.
The enzyme, alkaline phosphat<.~e is produced to sup-
plement their metabolism with organic c- rbon substrate.
which is also a final product of phosphoester hydrolysis.
Further the high phosphatase activity of bacteria is an
indication of high phosphorous flux in these microenvi-
ronments.
Effect OjLOlI1Telllperolllre on Memhrone COl1lpositioll
Plants, animals, and microorganisms predominantly
incorporate mono-unsaturated and polyunsaturated fatty
acids into the phospholipid fraction of their cell mem-
brane at low growth temperalLl res. Several detailed in-
vestigat ions have been undertaken to eluc idate the effect
of low temperatures Oil ratty acid unsaru rarion of mem-
brane lipids in psychrophiles. Mono- unsaturated and
polyunsaturated fatty acid containing li pids have been
implieatedmainly in the maintenance of liquid crystal-
line state of cell membranes. The modulati on of mem-
brane liquid crystalli,' <;(ate of its constituent lipids is
termed as homeoviscous adaptation. The phenomenon
is essential for the membrane to carry out its usual
cellu lar functions like solute transport. nutrient uptake,
assembly of transport proteins. and for the stahility or
membrane bound enzymes. The importance of a tem-
perature induced synthesis of desaturase has been re-
vealed in Bacillus Illegaterilllll. At 10w temperatures,
fatty acid synthase II acti vi ty increases several fold in
comparison to synthase I activity. Substantial amounts
of short-chain mono-unsaturated and di-unsaturated
fatty acid synthesis occurred in psychrophilic VilJrio
isolates grown at low temperatures. Another well known
PUFA 20:5 has been observed to be sy nthesized by a
marine Flexi/mcter spII. In one of the previous in vesti-
gations, several strains of Vihrio IIwrillllS grown at 2°C
were analysed for their PUFA's cOIll Position of ce ll
membranes. The sy nthesis of docohexaenoic acid
(22:6), and eicosapentaenoic acid (20:5) in these bacte-
ria has been found to oceur predominantly in response
to low growth temperatures. Bacterial strains cOlltaining
docohexaenoic acid displayed hi gher growth tempera-
ture but at less than 20°e. Whereas the strains with
eicosa pentaenoic acid ha ve di splayed a maximulll
growth between 20- 25°C (ref. 32). Ac ti ve synthesis or
PUFA's is a characterist ic feature of deep-sea microor-
ganisms. The synthesis of large amounts of di -unsatu-

5. RAMA 'A 1'1 (Ii.: BIOTECH OLOGICAL APPLICATION OF PSYCHROPHILES I) I
1~lted fatty acids ( 18:2) has been reported in Vihrio sp.
Evidence supporting this phenomena also has been pro-
vided by cold shock experiments in mesophiles as well
as in psychrophilic bacteria and yeast"·'~. Microorgan-
isms with high quantities of unsaturated fatty acids are
less susceptible to damage occurring at culture storage
temperature as low as minus ~m°c.
Cold-Shock Pm/eills ond Their Role in Cold
Tolerallce
The major inducible proteins such as cold-shock pro-
teins (Csps), cold accl imati on proteins (Caps), heat ac-
climation proteins (Haps) and heat shock proteins
(Hsps) are likely to pl ay an essential role in their cold
adaptation process.). The induction of cold-shock pro-
teins namely CspA. CspB. Cspc. CspD, and CspE has
been studied, in detail, mainly in Ecoli. These proteins
show amino acid sequence homology with each other"('.
T he cold- shock protein CspA (F IO.6) protein has been
designated as major cold-shock protein in bacteria due
to its high level of expression (200-fold increase) fol-
lowing a shift down from 37° to 10°C (ref. 36).The cold-
shock protein CspA is a 70 amino acid protein encoded
by the cspA gene. In the same famil y, there are other
genes responsible for the synthesis of cold-shock pro-
teins namely CspB.CspC, an I CspD with amino acid
residues of 7 1. 6lJ. and 74, respecti vely. Further these
proteins show 79. 70 and45 per cent seq uence homology
with CspA protein . The recentl y reported CspE protein
with 69 amino acid residues has shown 70 per cent
homology with the l1lajor cold-shock protein . Besides
the cold stress response in several mesophi Iic bacteria
has been shown to in volve the same gene products for
cell adaptation. Cold shock proteins, in general , have
been proved to elicit transcriptional and translational
control in prokaryotic as well as in eukaryotic organ-
isms. Illitiation of translation appears to be the most
thcnnoscnsiti ve step but elongation of the proteins can
occur uninterruptedly at low temperature as revealed by
Friedman el (fl.l7. IX . It has also been demonstrated that
the cell growth stops below 7.8°C onl y due to the
inabilit y to initiate protein synthesis. The induction of
co ld-shock proteins increases from 2 to 10 fold after
lowering the growth from 37° to 27°C'h These prote in ~
also include NusA which is required for termination and
anti- tennination of transcription. Initiation factor 2
binds to the charged tRN A'IlIC' to the initiation site 01"30S
ri bosomal subuni t for maintaining the translation proc-
ess. Its ability to combine with polynucleotide phospho-
rylase has been found to be necessary in the degradati on.
of mRNA .
In addition to these fact ors the role of trigger factor
(TF) in cold-shock response in mesophilic E coli has
also been described'H. This protein exhibits prolyl isolll-
erase activity and has been implicated in cold adaptati on
of bacteria. The expression of T F was found to occur ill
response to cold-shock suggesting that it isdistinct from
heat-shock protein repertoire. It shows affinity to GroEL
and enhances its binding to unfolded proteinsof the cell.
Based on these observations, presumably, TF plays a
major role in protein folding and also in the degradation
ofmisfolded polypeptides. Theexpression of the protein
was found to increase after initial cold-shock and during
storage of ELOli at 4°C. The pattern of expression
closely resembles with that of the well studied cold-
. ,I}
shock protem response' .
Protein Fo/ding and Inlrall/()/eclI/or Inler({ctiolls
Several factors have been described to contribute to
the fl exibility of enzymes in psychrophi les. Flexibil it y
of a protein conformation is often known to play a major
role in deciding the therlllosenisiti vity and catalytic
efficiency of enzymes. The ri gidity of a protein mole-
cules is mainl y reduced through changes in intramolecll-
lar interacti ons. According to recent in vest igati ons
protein surface properties like low hydrophobicity and
high hydrophilic contribute to the stabi lity of enzymes
in psychrophiles. Decrease in hydrophobicit y is a-;-
cribed to the presence of low number of proline residues.
However, increased hydrophilicit y is caused by the in-
corporation of morecharged amino acid residues to forlll
an extended surface loops. Nevertheless the decreased
content of isoleucine, arginine, and at times the total
content of arginine+lysine suggests that hydrophilicit y
can also be achieved by substitution of amino acid
'd h I I I d . . I ~() ~ I C .rest ues ot er t lan t l e c large amIno actcs . . ertal1l
psychrophilic enzymes are completely devoid of disul-
phide bonds, isalso attributed to the protein fl ex ihilit /'-
In spite of all these generalisations the data are insuffi-
cient to explain conclusively the role of various interac-
tions in enzy me stability in psychrophiles. Aspartare
transcarbamy lase produced by a psychrophilic strain of
antarctica di splayed 26 per cent or its optimum enzyme
acti vity at O°C~2. Some of the kinetic properties and
thermal stabi Iity of the enzyme resembled transcar-
bamylase of mesophilic Ecnli. It indicates that these
enzymes can achieve catalytic efficiency through spe-
cific changes in the catalytic site in add ition to c h ,lIl g('~
. ~UI
that occur In the enzyme molecule .

6. .I SCI IND RES VOL 59 FEBRUARY 2000
Further, diverse type of enzy mes were found to be
responsible for cold-adaptation apart from fatty acid
desaturases a~d cold shockproteins as well as cold ac-
climation proteins. was confi rmed by the transposon
mutant analysis. Goverde el of. ~'i, have demonstrated the
role of an exoribonuclease. polynucleotide phosphory-
lase (PNPase) in cold adaptation. The enzyme P Pase
mediates gene expression through the .degradation of
mRN A in the cell . Arter an initial cleavage by other
cndonucleol ytic enzymes the:r to 5' exonuclease acti v-
it y llf P Pase i:- responsible for the degradati on of
IllRNA in eubacteria . However, in E.co/i, two types of
:r to 5' exonucleases i.e. RNase II and PNPase, were
found to be responsible for exonuclease acti vity. A lso,
studies have revealed that the loss of thi s acti vity in
conditionall y lethal mutants was found to be detrimental
to the growth of E.co/i strain at low temperatures.
Ycrsilli({ elllemco/ili("(l a psychrotrophic isolate from a
transposon mutant library was found to depend on the
enhanced ex press ion of PNPase and its ml< A for eolcl-
adapt;lti on at 5°C. The cold inducible promoter con-
tai ned an ATTGG sequcnce characteri stic of co ld
proilluters~h Although inconclusi ve generalizati ons
ha ve been made by various aut hors some of the factors
which are responsihle for cold adaptation are: (i) Pres-
ence of suhstantia l amount of pol yunsaturated fatty ac-
id:- in the lipid Illoieties of cell membranes~7.4X, (ii )
Illodifications in enzy mc structure resulting in high spe-
ci fi c activity at low-temperatures i.e., low Kill and hi gh
,uhstrate turnover. This phenomenon is being reflected
in the activation energies of psychrophi Iic P.jll/orescells
which showed activation cnergy values of 10-38 .0
k.l /mol whereas the mesophilic protease showed 60.0
k.l/mol. (iii ) modifications in the D A replicati on, tran-
.. d I ' I . ,. I II ~I) 'iO
scnptlon. an trans atlon macl lnery 0 t le ce " .
Biotechnological Aspects
1 rC/IUe/ £n::.yllies os NOII('/ Bioc({lO/ysls
Several groups are actively engaged in the exploita-
tion of thermophilic ;!nd hyperthermophilic enzymes
from archae in developed countrics. Enzymes of ex tre-
l1lophiles known as ex trelllo7,Yllles can be empl oyed in
'ariou:- fields ranging from the production of bulk
chcmical:- to hea lthcare products. Ex tremozy mes can
optimally function at both cxtremes of temperature and
pH. On thc other hand. some microorganisms thri ve at
hi gh sa lt concentrations and, therefore. their enzymcs
exhibit ;1 natural tendency to work at high osmolality.
Thennm:Yllles refers to enzy mes which are produced by
thermophi les and hyperthermophiles. There enzymes
are optimally acti ve between 60- 125°C. The tempera-
ture characteristic of these enzymes makes them attrac-
tive in various biotechnological applications, They have
been used in molecular biology and one of the successfu l
sto'ries is attributed to DN'A polymerase of ThCrI1ll1S
aquaticlls and its wide use in po l y me ra~e chain reaction.
Detergents and starch hydrolysis for the product ion of
glucose and high fructose corn syru p using ~-amy l asc
and glucose isomerase respecti ve ly. Several of them
have been proposed for the synthes is of organic chemi-
cals in addition to diagnostic kits, waste treatment , pulp
and paper-milling and in the preparation of animal
feed'i. Thermostable enzymes have been shown to be
resistant to various chemicals and can be functional in
organic solvents in the absence of water, thereby leading
to many desirable properties. Proteases of Archae arc
known to hydrolyze highly res istant proteins like keratll1
for the production of amino acids . Alcohol dehydro-
genase produced by Th crllloallo cro/Jill/ll brockii is cur-
rently used in biosensors for the detection of alcohol ' !
and in the synthesis of chiral compounds" . The enzyme
is active at high solvent concentration and 6()°C. One of
the great advantages of using enzy mes in organic sol-
vents is to reverse the hydrolytic reaction occurring in
aqueous media. Enzymcs which hydrolyze esters, pep-
tides, and oligosaccharides in water can synthesize the
same compounds in organic solvents. Biosensors pn>
pared with thermophilic cn zyl1lc.~ do show rcmarkable
stability when exposed to various chemica l reagent:-
during immobi li zation procedure for biosensor and stor-
age during transportation in tropica l countries. Thermo-
stable enzymes may be used as antibody conjugates in
immunoassays, However the main constraint at prcsent
is their prohibitive cost of production and reluctance to
use innovati ve methods.
Hydrogenases are predominantly found in hyperther-
mophilic fermentati vc archae. In view of the depleting
energy reserves biophotol ysis of water by solar energy
has been investi gated to liberate hydrogen. Mel/wllo-
bacTeriulII IhernlOW/lOlroph iclIIlI produces hyd ro-
genase'i~. Immobilized enzy me processes based on these
enzy mes show an enormous operational stability mak -
ing a bioprocess cost-effecti ve. Higher yields of en-
zymes can be obtained with minimal loss of activit:
duri ng puri fication'i"'.

7. RAMANA £'1 (fl. BIOTECHNOLOGICAL APPLICATION OF PSYCHROPHILES 9]
Use of' Psychrophile Enzymes in Food Processing
Industrv
Psychrophilic microorganisms and thei r enzymes
have a wide range of applications in dairy and food
industry. Psychrophilic milk coagulating enzy mes have
the advantage of controlled casein coagulation for main-
taining the quality of whey resulting from cheese indus-
try which can be used in other processes. The enzyme
activity in whey can be destroyed by pasteurizati on.The
commercial microbial rennet available in the market in
developed countries with the brand names Marzyme,
Rennilase 50TL. and Moelilase are products of colel
active microorganisms. Another interesting application
of cold active enzymes is in the form of~-ga l actos idase.
Lactose hyd rolysis in milk and whey to galactose and
glucose results in increased solubility, digestibility, and
sweetness of milk. Usually, ~-galactosidase obtained
from mesophilic strains of Klu vermyces and Aspergillus
strains are acti ve at relati vely higher temperatures, i.e.,
~O-4()°C, and the milk has to be processed in conven-
tionalmethods for at least 4 hours for complete hydroly-
sis of lactose. These conditions increase the chances of
microbial contamination during the process. With the
use of thermolabile ~-ga l actos idases hydrolysis of lac-
tose can be carried out at 50
_10°C in about 16-24 h.
Using the cold active ~-galactosidase 70-80 per cent of
product yields can be obtained, which is much higher in
comparison to the processes obtained using enzy me
from mesophilic microorgan isms. A commerciall y
available galactosidase is in use for the purpose. The
commercial cold acti ve neutral protease is mainly ob-
tained from Bacillus suhrilis and being marketed under
the commercial name eutrase. The enzyme is known
to increase the fl avour intensity with reduction in the
ripening time from 4 to Imon.
Psychrophilic microorganisms are able to produce
various enzymes of industrial importance. Neutral pro-
teases from psychrophilic bacteria are being used in
cheese maturation. Polymer degrading enzymes such as
amylases, pullulanases, xylanases, and proteases are
employed in food processing. Proteases with low opti-
mum temperature and high pH are being marketed under
the commercial names Savinase, Maxacal, and Opti-
clean.
Source of' Norurul Piglllents
Carotenoids arc present in vari ous microorganisms
and they play an important role in protecting the photo-
synthetic machinery of the organ ism from photo-oxida-
tion. Several bacteria of antarctic origin can also pro-
duce pigments and mainly belong to the Flecw/Jacil-
lus5
1>, Pseudomonas".7, and Micrococcll.l''''x. As there is
growing tendency to use natural pigments, bacterial
pigments of different hues and colours may prove to be
handy and renewable source for food processing indus-
try.
Hydrolysare of'Biol11as.I'es As Feed Stock
The extracellular production of Lal11inaria sp decom-
posing enzymes were partly characteri zed in marine
bacterial isolates belonging to the genera AlreroJ11ol1us
sp, Pseudomonas sp, Moraxellu sp. and Flavo!Ja, "
teriun/'() These enzymes were found to be highly active
against several marine polysaccharides like alginate,
fucoidan, and cellulose. The bacteria population largely
consists of psychrophilic Vibrio sp whose optimulll
growth temperature ranges from 50
_20°C. In the deep-
sea, bacteria and cyanobacteria that take part in the
biodegradation of phytodetritus between 20
_ 15°C and 3t
a depth of> 4500 m60
1>1. The enzymes responsible for
decomposition were identified as alginase and fll-
coidanase at IS°C. Hydrolytic acti vity is a common
feature of marine bacterial populations as well as other
microorganisms. In future, this may help in producing
single cell protein and liquid fuel, after hydrolysis of
enormous amounts of sea-weeds and aquatic plant
biomass.
Several types of psychrophilic microalgae have been
reported from antarctica and other cold habitats. Be-
cause of their and inexpensi ve growth requirements
substrates comprising solar light and other inorganic
compounds present in marine waters can be used for
biochemical production such as vitam ins, carotenoids,
pigments polysaccharides, protein, and foods.
Lipids as Food Addirive.l'
Microbial lipids containing polyunsaturated fatty ac-
ids (PUFA's) are recommended to increase nutritional
value of food products and as additives in cosmetics and
as starting substrates for the preparation of pharmacell-
ticals(12 Polyunsaturated fatty ac ids are commonly
found in marine microorganisms
6
' . Probably these or-
ganisms produce PUFA's in response to low tempera-
ture of marine habitats.
Lipids extracted from psychrophiIic antarctica bacte-
ria and marine algae mainl y consist of CI 6 and CIS
unsaturated fatty acids. The marine algae AnadY()fIll'IIC'
.I'relLara can synthesize 16-22 carboo containing unsatll-
rated fatty acids possessing as much as four conjugated

8. lJ-l .I SCIIND RES VOL 59 FEBRUARY 200()
double bonds6
-1 . The synthesis and modification of fatty
acids mainl y occurs in chloroplast and in the endoplas-
mic reticulum of these eukaryotic microorganisms('''. A
group or psychrophil ic se;l-ice derived bacterial strains
;Ire known to produce polyunsaturated fatty acids such
; I ~ eicosapentaenoic ac id (20:5w3) and arachidonic acid
(20:4w6) . Bacteria belonging to the fam il y Flovo/mcle-
ri((ceu arc also known to synthesize a range of volatile
fatty acid containing lipids in addition to algae.
Sillg/(' Cell Prolein/i"()J1/ Chilill
Chi tinase (EC 3.2. 1.14) rrom Serratia marcescens
QMB 1466 was attempted to hydrolyse large quantities
or shell fi sh chi tin int0 solu hIe monomers such as N-ace-
ty lglucosamine and its conversion to singly cell protein .
The process includes size reduction, deproteination. and
demi ner;ili zation
llh
The hydrolysis or chitinmay also be
carried out using chitin frolTlmarine bacteria since chiti-
I
~ I " (,7
nase pro( ucers are preva ent In mari ne waters .
US£' oj' Hw /ro/vtif' £ 11;'.1'11/('.1' ill PII/p /ndllslry
Debleaching or paper mill pulp has extensively stud-
icd by many in vestigators. Arter cellulose xy lelll is the
most ahundant biopolymer in nature. The main sugar
cOlllponent or xy lan is D- xy lose. Heillicellulose con-
~ i ., t ~ or a .~e ri es or Ill.:teropolyselccharidcs that .include
glucans. ma nnans. arabinans, and xy lans. Microbial en-
zylllL:S dcgrad ing hClll iceliuloscs have great industri al
applications. Cell ulase frec heillicell ulases arc userul in
the pu lp and paper industry ror bleaching of krart
plll p('~'(,'} Xylanases are usefu l in the modification or
pulp during paper making anel 1'0 1' recycling of waste
paper. Wi th the eliscovcry or morc andlllore varieties or
hL:llliccliulases it would be possible to apply them com-
Illercially in the pulp industry. Alka line xy lanascs arc
pan icul;II'ly prererrcd in pu lp 1ll;lking proccss or p;lper
industry. Incrcasing solubilit y or xy lan at hi gh pH helps
in incrcasing thc hydrolys is or xy lan by thcse enzy me.
Xylanascs arc large ly produced by alka liphiles or thc
genu s 13((cillllS. Alka lophi le /J{/ cillllS strai ns opti ma lly
grow at hi gh pH and their xy lanases also display opti-
IllUIll acti vity at pH > 9.0. B{/cillllS .l'Ifhlili.l' xylanase
hydrolyses lip to 3X.Oper ccnt or thc substrate with low
produ c t inhibit io n co nst;In ts in co mpari son to
l'rich()r/£,J'/I/({ virir/{/(' xylanse preparation. However. xy-
lanase pruduct inn by pscy rophiles has not been at-
te':ll pted. Some or the ;il ka liphilic /JucillllS sp xylanase
showed higher opt illlu l1l pH and temperature (60°C). In
spi tl' or all these developillents xy lanases or thermo-
philic alka liphiles ;Ire prefe rable to wit hstand hi gh pul p
temperature of 60°C and pH > 9.0. Zakaria el u/
n
have
reported the synthesis of ~- man nana~e in a psychro-
philie strain of F /a vo /)({Cleri /lll/. The enzyme synthesis
occu rs in the presence or I per cent guar gum and it wa~
able to hydrolyse efricientl y the same substrate opti-
mall y at 35°C. At 10°C the enzyme di splayed 25 percent
of its optimal acti vity.
Microorganisms producing a8etyl esterases which
are functionally and physiologically similar to lipases III
combination with u-g lu co u ronid ase~ are required in the
pulp making process to obtain max imulTI xy lelll hydroly-
sis. Bio-pulping/enzymatic pulping for xylan h ydro l ysi ~
and cellulose deri vatization also requires the cflopera-
tive activity or es t e ra~es in the enzymatic degradation or
70
acetyl xy lans .
Use oj' £ n;,vllle.l' ill Biodelergenl.l'
The concept of utili zing enzymes ~t arted in 19 13 by
Otto Rohm has developed now into a major commercial
exploitati on of proteases, li pases, amylases, and cell u-
lases as detergent add it ives71. Remarkably, most of these
enzy mes are being produced rrom microbia l sources
including bacteria, yeast. and fungus or genetically cn-
gineeredll1icroorganisms. These en7.ymes arc marketed
at present throughout worl d under seve ral bra nd names.
Thesc enzymes arc optill1all y acti ve between pH 9.0-
11 .0. Genetic engineering is widely used to increase
enzyme yields from wild microbial strains which arc
uneconorl1ical for large-scale production. Subti lisin type
of proteases and u - amy lases ha ve been round to he
suitable for detergent industry. Alpha amylases arc use-
ful not only as add itives of laundry detergents hut they
are incorporated into powder and Iiqu icl formul ations 1'0 1'
industrial andmachinc dish-washing.
Protease, li pase, aillylase, and cL:liulases have been
widel y reported in psychrophilic microorganisms and
they Illay prove very usefu l 1'0 1' low temperature wash-
ing. The principle of the so called "greener" and milder
chemicals detergents is based on enzymes di splaying
hi gh activity at low temperatures. Cold washing telll-
peratures are preferred in several countries. Amylases
and other enzy mes which arc stable in the presence or
protease prove to be preferable 1'0 1' kitchen ware wash-
ing. With the discovery or novel enzymes in pscym-
philic, in rUtll rC, it will be p()s ~ i b l c to rorm ulatc
detergents 1'0 1' vari ous purposes. Ps)'c hrophil ic ~li b l ill e
p rot ea se~ report ed in thc'antarctic P.I'('{f{/()" {()IIU.I' sp and
AC/'{J,,{()lIu.I'·Ityrlmphi/u strain can he utilized a~ deter-
gent add itive
2x
. Enzy mes with 1ll;lx imull1 activity at

9. RAMANA ('I o/.: BIOTECHNOLOGICAL APPLICATION OF PSYCHROPH ILES 95
alkaline pH and stable at moderately higher tempera-
tures are hi ghl y useful as detergent additives:12
Psychrophiles As A SOllrce of' Pharnwceuticals
Discovery of secondary metabolites from marine mi-
croorganisms hJS been drawing much attention for the
last three decades. As a consequence, several strains of
bacteria, streptomyces, and fungus have been reported
to produce antifungal, anticancer,and anti-tumor agents.
It is evident that most of these microorganisms belong
to the category of psychrophi les as they have been
isolated from deep-sea sediments, marine waters and gut
flora of aquatic an imals and plants72
The bacterial gen-
era that produce these agents are: Streptol1lvces, Altero-
/1/011£1,1'. [3u cilllls. Mic{'ococc l/s . Ae{'ol17o l1 as.
Fhll'o/)ucleriulIl. Moru..rel/o, Pseudoll1o//as, and Vihrio
with growth temperature between -3 to 3()°e. Aquatic
plants and animals are highl y prone to infestation by
pathogenic microorganisms. Surface bacterial symbio-
sis is an essential adaptive and protective mechan ism in
fresh water and marine sed iments. An Alterol11olws 'iP
has been reported to synthesize 2,3-indolinedione (isa-
tin ). Experiments have amply demonstrated that this
compound protects the shrimp Palaell1o/7 macrodact,'-
IllS from a pathogenic fungu s L(/genidilllll ccrLLillectes.
Similarly. another strain of AlterolllO//as sp, intimately
associateci with the marine sponge Holichondria okac/o.
prod uces a tetracyclic alkaloid namely alterimide A T".
There is a wide scope for the discovery of novel biologi-
call y active cOlllpounds in marine microorganisms. By
learning their structural intricacies the development of
new pharmaceutical prociucts might be possible in fu-
ture. Studies have also indicated that marine strains of
Morctxel/({ sp ancl Flol'olxlcleril/lI1 sp can produce anti-
. I I . . I 74 7S TI .vlra ane anti-tumor agents. respecti ve y " . le anti-
tumor polysaccharide has been iclentified as narinactin .
Pathirana et (117
(, isolated L-quinovose class of esters
from marine actinolllycetes. Unfortunately, biologically
active compounds from marine microorganisms have
not been exploited. Jensen and Fenical have adequately
emphasized on the potential of marine microorgan-
isms
77
Similarly. antarctic bacterial isolates may yield
several of these metabolites. since most of the genera
also occur predominantl y in antarctic habitat. To our
knowledge, so far no attempt has been made to screen
antarctica these bacteria for the production of biologi-
cally active compounds.
Shaver and Schi ff et (/1. 7K have demonstrated the
usefu lness of protease and amylase mi xture from Bacil-
IllS slIhtilis in removing the dental plaque. Lipases are
mainly used as stereo-specific catal ysts and in the
biotransfonnations of various high value compouncis
I tOI ' d I . I 7')-X I
SUC 1 as avounng agents an p larmaceutlca s .
Concentrates of polyunsaturated fatty acids of the type
3-3 are obtained by the hyd rolysis of fish oill ". Fatty
acids such as gadoleic acid, erucic. and nervoni c acids
are obtained by the hydrolysis of seed oilsX:1. Lipolytic
enzymes are widely employed for the extraction of these
high value nutropharmaceutics. Lipases have been stud-
ied mainly from antarctic Moraxel/a 5. 17, Pseudomo/7(/s
and Bacillus sp and Serratia liquefaciens isolated frorn
blue green algal matsK-l. Lipases from psychrophil ic
bacteria are preferred because of their unique positionJI
specifity for fatty acids not found in mesophilic en-
zymes. The enzy me frolll a psychrotrophic Pselldo-
1I10nas strain was found to ex hi bit 1,3- positional
specificity towards triglyceride substrates like triolein.
Owing to their low activation energies, i.e. 11 .2 and 7.7
kcailmol, cold-acti ve lipases are catalytically more effi-
cient at low temperatures in comparison to mesophil ic
enzymes. The enzyme was found to be activated by
organic solvents like methanol and dimethyl sulphoxide
from 0-30 per cent (vol/vol). Enzymes which show
stability in organic solvents are biotechnologicall y im-
portant to carry out enzymatic reactions in organic sol-
vents. This aspect of biotransformation is gaining great
importance to increase reaction rate and to extend the
substrate specificity ofenzymes. Use of organic solvents
as a medium of reaction aids in solubilising the COI11-
pounds which are insoluble in water. In the bioconver-
sion of water insoluble compounds such enzymes arc
quite useful. Bacteria able to grow in the presence of
high concentrations of organic solvents have been re-
ported from deep-sea sediments with an optimulTl
growth temperature of lODe. They have been ident ified
as genera Flavobacterium. Bacil/lls sp, and Arthro/Ju( '-
tel' and can withstand high concentrations of benzene.
toluene, and p- xylene8S
Recently, it has been found that
immobilized enzymes in organic solvents are efficient
in terms of their velocity to catalyse biochemical trans-
formation to obtain biologically active compounds.
Trehalose (u-D-giucopyranosy I u-D- glucopyra-
noside) is a non-reducing disaccharide widespread
among bacteria. The sugar is economicall y important
because of its function as cryoprotectant to stabilize
proteins and protection of animal tissues. The sugar
finds various applications such as sweetener, stabilizer
of frozen foods, in cosmetics and as a drug additi ve.
Trehalose is formed by an enzyme trehalase present in

10. ()h J SCI I 0 RES VOL 59 r EBRUARY 2000
several psychrophilic and thermophilic bacteria. Psy-
chroph iIic microorgan isms synt hesize the sugar to pro-
tect the cell rrom desiccation. heat, and osmotic shock.
USCS III'Buclerio/ 1('(' NlIdeul ill~ ;~ellis
There arc seve ral uses ror ice-nucleating agents
(I A) produced by bacteria. They are being used in
artiricial snow making, in the production or ice cream:-.
and ot her frozen foods. In immunodiagnostic kits as a
conjugate to antibod ies and as a substitute 1'0 1' silver
iodide in cloud seeding. Among several organisms, rNA
from bacteria have attracted Illuch prominence OWIll ~ !O
theiI' abi Iity to form ice nuclei at relat ivel y high templ.:l a-
lUre in cOlllpari son to other sou rces. The subject has been
reviewed in detail hy Lindow ('I u/.X6
.
X7
PselldolllOrws
"."ri,lgl/{'. /'SCIf(/o/IIOIIUS .fI//oreS(·ells. PS(!IIdol1lOlf(lS
I'iridijl(/'{/. E/''illi(/ herhic()/u. and X(/I7I//()IIWIWS C(flll -
/1('slri. are the well known producers or INA. Further,
:-,ollle marine strain:-. of PsclIdllll/()lIusjlllo r(!scells have
been ~ h own to produce IN As. Most or the bacterial
strain~ u~ed ror producing INA are psychrophilic in
flatu re. Severa I properties or INA reselllble psychro-
phi lie enzymes. Particularly. I A~ are thermolabi le
Illolec ule:-.. For many practical uses, ice nucleati ng
agents rrom bacterial sources are preferable, since mi-
croorganisms can he illlProved by recombinant D A
techno logy and they can he mass produced in bior'eac-
tor:-.. The unique property or hacterial ice-nucleating
age nt~ is that they arc least soluhleeven at hi gh tempera-
lure.~ in comparison to plant and insect agentsxx. An
optilllum growt h temperature or2{)OC is required 1'01' the
:--ynthesis or INA in hacteria such as P.svrillgo(!. The icc
ilucleating temperature as well as the synthesis or 1 A
is highly influenced by h,lcterial growth temperature.
INA produced at 2()OC preserve the propert y or icc
nuc leati ng activi ty at higher temperature. In the case
1'J!lIorc.·('el1.·x'l. l 's{,lIdolll()1I0S s,'/'illg(/c is an 0 th eI'
widely occurring hacteri,iI epiphyte. The IN A is pro-
duced in the rorm or an outer membrane protein. Large
aggregates or I A can orient water molecules into crys-
t,illattice mimicking the nat ural ice particles and leading
to a cascade or ice crystal rorlllation
xh
US(' ill F enll(!lIll1lioll Indllsll''
Me:-.ophilic yeasts which contain unsaturated ratty
,Icitis in membranous lipids hilve been round to be resis-
tant between -~m to -2{) dc. normall y prefe rred for their
~torage in baking :Jnd other rood processing industries·JI
,.
I-or ine production, rerment ation at 6-HuC has been
round tn reduct' the inhibitory errcct oj ethanol 011 cell
meillbrane of yeast cells'll. Specifically, cryotolerant
yeast strains have been selectively obtained. Sparkling
wine is produced Llsing the yeast strains which .arc
tolerant to hi gh alcohol concentrations.
Applicatioll ill Micm/Jio/ L eochillg
Currentillicrobialleaching operati ons involve ox ida-
ti ve solubi Iization orcopper and uran iUIll ores. Leaching:
operations in teillperate cOllntries have to be carried out
at very low aillbient temperature. The temperature de-
pendent variations of microbial acti vity has been noticed
to affect the ore leaching operations by Ahonen and
Tuovinan'12. Microbial leaching operation from sulphide
ores was studied at low temperature between 4°-:n°c.
Probably the process in volves psychrophilic microor-
ganisms and illlprovellleills in this area wou ld be possi-
ble by empl oying pure cu ltures or the bacteria or
antarctica origin or from other cold habitats.
Usc ill Biorel71ediclliol1
In add ition to their abi Iity to bi od e~ rad e various CO Ill-
pounds in the habitat they can be proillising in biorellle-
diation or several pollutants at low-temperatures. The
occurrence ofbiodegradative psychrophiles is relatively
more in contaillinated sites where the aillbient tempera-
ture overlaps with the optilllum growth temperature or
these microorganisms. Psychrotrophic hydrocarhon ck-
grad ing bacteria have been isolated rrolll oiI poll uted
sites or Ontario and Quebec and it ha:-. been noticed that
the occurrence of hydrocarbon degradi ng Illicroorgan-
iSllls is low from relatively pristine environmental habi-
tats'll. The bacterial strains were found to Illinerali ze
dodecane, hexadecane. naphthalene, and toluene at 10
temperatures and it has been evident in laboratory and
fi eld experiments, utilizing specific bacterial cultures.
Some of the degradati ve genes responsible 1'01' catabo-
lism or naphthalene (ndoB) and toluene (xy IE. todC I)
have also been detected in psychrotrophic bacteria using
molecular techniques sllch ;IS southern hyhridi zatinn
and polYlllerase chain reactiun'I-I. P:-.yc hrophilic ba-:teria
that can degrade high Illolecular we ight hydrocarbons at
low temperature were described earl ier'I'.
A psychrotrophic bacteriulll, Rhodo('()cclI.I sp. stralll
Q 15 has been studied ror its ability to degrade n-alkane~
and diesel fuel at low temperature. The strain mineralize
short chai n alkanes li ke dodecanc anci hexadecane .It
hi gher rates than the long chain hydrocarhons such ;1:-'
octacosane and dotriacontane (()"'-,'i°Cl. Also the ~l raln
has been able to Ill llleralize at hoth branched a lkan e~ and
substituted cyclohexalle~ PI'L'sl'nt iL di c~el (lil () (' ) III

11. R!M ANA 1'/ fit. rl lOTECHNOLOGICAL APPLICATION OF PSYCHROPH ILES
addition. minerali zati on of hexadecane at SoC was no-
ticed in hydrocarbon contaminated and pristine soil
. . I . R/ / '!6
microcosms uSing tle organis m IO{ OCOCCIIS . The
psyc hrotrophic bacterium Rhor/ococclIs sp. strain Q 15
that can degrades n-alkanes and diesel fuel at ~o_S oC
was shown to contain the gene responsible for the pro-
duction of al iphatic aldehyde dehydrogenase with DNA
sequence resembling the mesophilic R.eryfhmpolis
thcA genc.
il7aemlJic Digesfio/l o(Orgonic W(fSfeS
The obligate anaerobes which convert organic acids
to CH.j and CO2 (methanogens) are highly sensiti ve to
low temperature and pH levels. Psyc hrophilic anaerobic
bacteria are not extensi ve ly in vestigated in comparison
to aerobic bacteria. owing to their very low growth rates
and cumbersome isolation procedures. However, there
are some reports that describe the isolation of psychro-
philic anaerobi c bacteria from antarctica habitat. Vari-
ety of organic wastes can be degraded to innocuous
prod ucts by an,:erobic degradat ion. It is one of the
potential routes for methane generation in cold habitats.
Further. anaerobic di gesti on has assumed much impor-
tance in the treatment of vari ous tox ic chemicals and
hi gh stn~n gth industrial wastes. Isolation of psychro-
philic anaerobic microorganisms is of immense impor-
tance to supplement the anaerobic digesti on process to
he carried out at low-temperatures. At present, Llllaero-
bic digestion of sewage sludge, cow dung, and other
animal wastes is treated by adapted mixed microbial
. 'J7 . .
consortia . Nevertheless, microbial growth and meth-
ane production is ve ry limi ted at sub-ambient tempera-
tures (~S O C). The rate of methanogenesis can be
Increased several fold by low temperatures adaptation
of methanogens The process can be made possible by
selecti ve enrichment of psyc hroph iIic methanogens
through long term laboratory trials. Hence the basic
understanding of these microorgan isms is essential for
improving the performance of the methanogenic proc-
esses. Besides methanogens. bacteria with the abi lity to
reduce sulphate have been isolated and identified as
t) N
/)e.w/(orhopa/lls "({CIIO/{flllS ' . Few reports have re-
vealed the importance and the role of methanogens in
antarctic habitat. In one of these studies, a psychrophi Iic
strain of M ef//{/l7ogenilllll/i'igidlllll sp nov., was isolated
from Ace lake, antarctica which grows optimally at
Isoc. The organism was found to produce methane from
hydrogen and carbon diox ide. The growth rate of psy-
chrophilic methanogens is very low (0.24 d·l)with a td
of 2.9d()(). Simi larly, an acetoclastic psychrophi Iic strai n
of M ethwwcoccoides /Jur{ol1i from Ace lake, antarctica.
has been reportedJ(X).
D el1.itrijicofiol1 af Drinking Water Sources
The presence of hi gh nitrate concentrations in water
has been a major problem in many countries includillp:
the US, Canada, and Eu rope. Drinking'water containinp:
0 ,- quantities exceeding 10-50 mg/l are harmful. The
most widely used practice for the removal of NO- is
biological denitrification. Most of the denitri fi cati on
processes are carried out at about 'Wc. HolI () and
C k lUI I d ' b' I I .. ,.. .za 0 , eva uLlte micro ILl CenItn Icatlon process at
12°C and the maximum specific nitrate removal rate
observed was n o mg NO -/h/g cells. The nitrate re-
mova l capacity of the reactor at I(j°C was SO to 60 kg
NO,-/m' /d. To increase the treatment effi ciency at these
temperatures, immobilized microbial cell based biorc-
actors such as packed bed, and fluidi zed bed are being
used. Also, a fu ll scale denitrification unit using the
same process has been operated between ' 20
and 18°C.
Temperature decrease of few degrees may redllce the
process effi ciency substant wll y which emphasises thc
need to use cold acti ve deni trifying bacteria. The deni-
tri fication efficiency has been found to reduce hy abollt
~3 per cent while the maximum denitrification rate ~It
18°C recorded was 2.24kg NO,-/m' lcl'll . However. re-
port does not conform to the presence of psychrophil ic
nature of these microorganisms. However, it appears
that microbial population consists of cold-adapted
mesophi Iic bacteria funct ion ing at lower temperature
probably with lower denitrification rates. The rate or
denitrification in these cases can be enhanced hy em-
ploying psyc hroph il ic bacteria isolated from perma-
nentl y cold habitats.
Biological Amendment of Psychrophiles and
Genetic Engineering
With the advent of polymerase chain reaction, vari-
ous genes responsible for biodegradation and metabo-
lism ha ve been identified and characteri zed from
psyc hrophiles. Recombinant D A tec hniqu e.~ have
been proved to be of great utility in the elucidation of
properties of crenarchaeota which cannot be culti vated
under laboratory conditions. Two subtilisins prod uced
by a marine isolate of Bacillus T A3Y and TA4 1 strain
have been purified and the genes responsible for their
synthesis are identified as Subt I and subt2. The enzyme
displayed a high catalytic efficiency at low and moder-
ate temperatures. However, the enzyme was highly ther-

12. .I SCII ND RES VOL)<) FEBR UARY ::W(){)
mosensiti ve as revealed by its flexibility and 3-D protein
conrormation analys is. The gene responsible for a-amy-
lase production in a psychroph iIic strain of A /lemmol1({s
ha/op/all clis has been cloned in mesophilic E.coli to
probe its protein rolding mechanism and effect host cell
physiology on enzy me acti v it l~. Studies have shown
that the enzyme expressed was simi lar in every aspect.
T he heterologous expression system provides a means
ror large-scale producti on or cold acti ve enzy mes in a
cost-effecti ve manner. T he erfect of intramolecular in-
teractions that increase the ri gidity of the protein Ll:lck
bone have been inserted by sit e directed mutage n esi~ , d
asses~ the impact or flexibility on thermosensiti vity or
the enzy me. T he ri gidity has been increased by creating
additi onal salt-bridges, aromatic interactions and by in-
. I 'f'" f l ' . I(P- I()~
creaslIlg t le a1 Inlty or ca clum Ions - .
Extremozymes or psychrophiles can be mass pro-
duced without growing the actual culture. Gene cloning
or psychrophilic enzymes may enable them to produce
in pu re rorm by modern met hods or protein ex pression
ill heterologous hosts such as I:'. coli using simple med i;i
components. M ost often it is dirficul t to rind an enzy me
wit h propert ies matching with the react ion conditions.
Thererore, to mod iry the enzyme for a specific task
bio-amendment or rational design has been carried out.
As a result, xerozyrnes or enzymes roreign to the nature
have been created using chelllical and protein engineer-
. I ' 10:;
Ing teclnlqucs .
Mesophilic bacteria can ex press enzymes of psychro-
philic bacteri a without drastic change in their catalyt ic
prnperties. It has been shown that cioning or Iipasc genes
illt o mcsophi lic. E.co/i pr ' :-,ervcd its low temperature
acti vity and thermolabile property or cold acti ve bacte-
ria. Hcterologous ex pression or genes rcsponsible 1'0 1'
cold act ive enzymes in mesoph iles. thcrerore, offers a
convenient means or econom ical producti on of cold
. I I ' . I I' . 10(,
enzymes In arge-sca e tor commercia app IcatlOns .
On the same ground. over expn::ssion of a psycl1r()phiIic
(X- amy lase gene was delllonst r;lted in a mesophilic
strain or E.Coli. The st ructural inve. tigations revealed
that the ]-D conronnalions and activity or the enzymc
were :-.imilar with thc wild type enzyme. For the produc-
tiOIl or enzyme" hetero logoLl," expre:-,:-,ion systems such
a, in I:'.r'oli. an opt imum tcmperature has to be deter-
mined in order to obta illmaximum growth orthe organ-
i."1ll <1I111 also 10 I11d lnlaill st"bil ity or the ex pressed
cll/.ymc. T illiS. isolation or nove I p:-.ychroph iIic micro-
orga ni ,~ ms would widen the gene diversi ty required for
oht;li nin!,! usefu l chcmicals through recombinant DNA
technology. M ethods like site-directed mutagenes is and
protein engineering, have been followed to increase thc
catalytic efficiency or enzymes at ex tremes of tempera-
ture, pH, and in organic solvents. Crellarc/welll1l .1'.1'111-
hiosulI1 an uncult ivated psychrophi Iic microorganism
belonging to the kingdom Crenarchaeota has been re-
ported by Schleper el a/
1
°. Culturing or thi s microorgan-
ism under laboratory conditions has not been pos. iblc
and it s phenotypic characterizati on was carried out
solely using molecular techniqucs and also dependi ng
on it: distribution in cold and temperate habitats. After
isolation and ampliricati on of DNA and RNA. the ge-
netic material has been ex pressed in mesoph iIic host:-,
for elucidating properties of their gene products. The
in vestigations carried out under similar lines, revealed
the presence of a DNA polymerase in C. svlllbioSIIIJI and
it was found to be thermolabiIe with rapid loss or acti vity
at 4()°C.
Protein engineering ha:-, been most IIseful in under-
standing the complex relationship between protell1
structu re and runction al low temperat tlrt'. T he techniquc
has orfered an effecti ve means to understand and prcdict
the contribution of covalent and ionic interact ions for
protein stability, flex ibility, and enzyme act ivity.
A spartate carbamoy l transferase hus been studied
relat ively more with respect to its molecul ar :-.tructure
and enzymology, since th is is one or the mu ltimeric.
allosteric enzyme prcsent in psychrophi Iie, as well a:-,
psyehrot rophic bacteria. Attempts have been made t()
unravel the changes that could occur in enzy me activity
and controlling mechani sm after its clnning in heterolo-
gous expression systcms. The genes responsihle ror
ATCase in a Vihrio st rain 269] hilvc heen clolled III
mesophilic E.co/i stra in')~ .
Conclusion
T he application or ex trel11ophiles, in general, anci
psyc hrophiles, in particular. in industrial processc:-,
opens up new era in biotechnology. Each group has
unique biochemical realUres which can be exploited for
use in biotechnological industri c:-.. The main rC;I:-,on ror
select ing enzy mes rrom thi:-, group of lI1icroorgalli slll:-' i:-,
their high stabi lity and reduced risk or contaminati(in at
low temperalUrcs. Due to thei r UllllSWtl properties thc"c
enzy mcs are expected to fill the gap bctween hiological
and chem ical processes. However the biotechnology or
extrel110plliles is sti ll in its inrancy but ha~ important and
rar reaching implicat ion.

13. RAMAN A 1'1 (I/.: BIOTECHNOLOG ICAL APPLICATION OF PSYCHROPHILES lili
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