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ScienceDirect
Current developments in marine microbiology:
high-pressure biotechnology and the genetic
engineering of piezophiles
Yu Zhang1, Xuegong Li2, Douglas H Bartlett3 and Xiang Xiao4
A key aspect of marine environments is elevated pressure; for
example, 70% of the ocean is at a pressure of at least
38 MPa. Many types of Bacteria and Archaea reside under
these high pressures, which drive oceanic biogeochemical
cycles and catalyze reactions among rocks, sediments and
fluids. Most marine prokaryotes are classified as piezotolerant
or as (obligate)-piezophiles with few cultivated relatives. The
biochemistry and physiology of these organisms are largely
unknown. Recently, high-pressure cultivation technology has
been combined with omics and DNA recombination
methodologies to examine the physiology of piezophilic marine
microorganisms. We are now beginning to understand the
adaptive mechanisms of these organisms, along with their
ecological functions and evolutionary processes. This
knowledge is leading to the further development of high-
pressure-based biotechnology.
Addresses
1
State Key Laboratory of Ocean Engineering, Shanghai Jiao Tong
University, Shanghai 200240, China
2
Deep-Sea Microbial Cell Biology, Department of Deep Sea Science,
Sanya Institute of Deep-Sea Science and Engineering, Chinese
Academy of Sciences, 62 Fenghuang Road, Sanya 572000, China
3
Center for Marine Biotechnology and Biomedicine, Scripps Institution
of Oceanography, University of California, San Diego, La Jolla, CA, USA
4
State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong
University, Shanghai 200240, China
Corresponding author: Xiao, Xiang (xoxiang@sjtu.edu.cn)
Current Opinion in Biotechnology 2015, 33:157–164
This review comes from a themed issue on Environmental
biotechnology
Edited by Spiros N Agathos and Nico Boon
For a complete overview see the Issue and the Editorial
Available online 13th March 2015
http://dx.doi.org/10.1016/j.copbio.2015.02.013
0958-1669/# 2015 Elsevier Ltd. All rights reserved.
Introduction
Marine ecosystems have always been a fascinating topic
for scientists. A heroic era of deep-sea research began the
moment the HMS Challenger weighed anchor. From
1872 to 1876, the Challenger Expedition explored the
Mariana Trench and many prodigious life forms at great
ocean depths [1]. In general, the geological settings of the
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deep ocean cannot be described in terms of their physical
and chemical characteristics, except with respect to the
high hydrostatic pressure (HHP). ‘Barophilic’ was the first
term used to define ‘pressure-loving’ organisms [2] but
was subsequently replaced by the term ‘piezophilic’
(from the Greek ‘piezo’, meaning pressure) [3]. The
current terminology defines pressure-adapted microor-
ganisms as piezotolerant (with similar growth rates at
atmospheric pressure and high pressure), piezophilic
(with more rapid growth at high pressure than at atmo-
spheric pressure) or hyperpiezophilic (with growth only at
high pressure) [4,5]. The pressure maxima increase in
rank order, which reach the peak point for hyperpiezo-
philes. Organisms that grow best at atmospheric pressure
and with little to no growth at increased pressure are
termed piezosensitive. As sampling and cultivation tech-
niques have been largely improved, it has become possi-
ble to isolate various piezophilic microorganisms from the
deep-sea, including certain obligate piezophilic species
that cannot grow at atmospheric pressure [6,7]. In this
review, we focus on how HHP has profoundly influenced
the life strategy of marine microorganisms. We also de-
scribe the primary research strategies used by marine
microbiologists to study these organisms.
Piezophilic microorganisms and HHP
adaptation
Since the first known obligate piezophile, Colwellia sp.
MT-41, was isolated from the bottom of the Mariana
Trench by Yayanos and colleagues [8,9], many other
isolates have been obtained from the deep ocean, hydro-
thermal vents and sub-seafloor (Figure 1). Most of these
piezophiles are psychrophilic gram-negative bacterial spe-
cies that belong to the Gammaproteobacteria class, which
includes species from the genera Shewanella, Psychromonas,
Photobacterium, Colwellia, Thioprofundum and Moritella [5].
Few piezophilic bacteria belong to the Alphaproteobacteria
and Deltaproteobacteria classes. All of the piezophilic
archaea that have been isolated thus far are thermophilic
and fall within the Euryarchaea and Crenarchaea king-
doms, including the obligate piezophilic anaerobic hyper-
thermophilic archaeon Pyrococcus yayanosii CH1 [10,11].
By obtaining an increasing amount of complete genomic
and metagenomic data, we are beginning to better un-
derstand the nature of piezophiles. For example, deep
ocean microbial assemblages tend to be characterized by
larger genome sizes, longer intergenetic regions and
Current Opinion in Biotechnology 2015, 33:157–164
158 Environmental biotechnology

Figure 1

74 Shewanella benthica DB172R (6499 m)

51 Shewanella benthica DB5501 (2485 m)
83 Shewanella benthica DB172F (6499 m)

85 Shewanella benthica DB6101 (5110 m)

100
Shewanella benthica DB21MT-2 (10898 m)
50 Shewanella violacea DSS12 (5110 m)

93 Shewanella piezotolerans WP3 (1914 m)

99 Moritella abyssi 2693 (2815 m)
Moritella sp. PE36 (3584 m)
79 100
Moritella profunda 2674 (2815 m)
52
Moritella yayanosii DB21MT-5 (10898 m)
64 Moritella japonica DSK1(6356 m)
Photobacterium phosphoreum ANT -2200 (2200 m)
65
100
Photobacterium profundum DSJ4 (5110 m)
Photobacterium profundum SS9 (2551 m)

Bacteria
100
Psychromonas hadalis K41G (7542 m)
87 Psychromonas sp. CNPT3 (5800 m)
100
78
Psychromonas kaikoae JT7304 (7434 m)
99 99 Psychromonas profunda 2825 (2770 m)
Colwellia piezophila Y223G (6278 m)
91 100 Colwellia sp. KT27 (9856 m)
Profundimonas piezophila YC1 (6000 m)
87
Thioprofundum lithotrophicum 106 (3626 m)
Desulfovibrio hydrothermalis AM13 (2600 m)
79 Desulfovibrio piezophilus C1TLV30 (1693 m)
100
100 Desulfovibrio profundus 500-1 (500 m)
56 Piezobacter thermophilus 108 (3626 m)
100 Rhodobacterales bacterium PRT1 (8350 m)
76 Carnobacterium sp. AT7 (2500 m)
100 100 Carnobacterium sp. AT12 (2500 m)
Dermacoccus abyssi MT1.1 (10898 m)
Marinitoga piezophila KA3 (2630 m)
98 Methanocaldococcus jannaschii (2610 m)
100 Methanocaldococcus fervens (2600 m)
Methanocaldococcus vulcanius (2600 m)
100
Methanopyrus kandleri (2415 m)
Palaeococcus ferrophilus (1338 m)
Archaea
83
62 Palaeococcus pacificus DY20341 (2737 m)
90 Thermococcus barophilus MP (3550 m)
100
Thermococcus peptonophilus
98 Pyrococcus abyssi GE5 (2200 m)
97
Pyrococcus horikoshii OT3 (1395 m)
50 Pyrococcus yayanosii CH1 (4100 m)
85 Pyrococcus glycovorans AL585 (2650m)
Pichia guilliermondii
100 Meyerozyma guilliermondii

0.1
Current Opinion in Biotechnology

Phylogenetic tree constructed on the basis of 16S rRNA gene sequences, showing the distribution of piezophiles. Tree topography and
evolutionary distances were determined using the neighbor-joining method with 1000 replicates of bootstrapping. Bootstrap values providing
50% support are indicated. The scale bar indicates 0.1 substitutions per nucleotide position. A red star indicates species with constructed
genetic systems; a black triangle indicates species containing plasmid(s); obligate piezophiles are shown in bold; water depths of the original
samples are indicated in brackets.

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 HHP review Zhang et al. 159

Box 1 A general decrease in temperature and increase in
oxidation in the natural habitat of early life
Immediately after the ocean formed and the asteroid impacts that
caused extinction stopped, life originated on the Earth’s surface —
the Precambrian ocean. The oxygen isotopic data from early
diagenetic charts indicate that the surface temperature of the
Precambrian ocean was between 55 8C and 85 8C throughout the
Archean period [51]. Oxygenic photosynthetic bacteria appeared on
Earth 2.7 billion years ago, and oxygenic photosynthesis promoted
the oxidation of the early atmosphere only 2.4 billion years ago
[52,53]. Thus, to become the modern Earth’s surface, there was an
inevitable cooling and gradual oxidation process.
higher ratios of rRNA operon copies to genome size than
their surface counterparts [12–15]. No novel mechanism
for HHP adaptation has been found thus far, although
HHP stress-induced proteins are similar to those induced
by other environmental stresses, for example, tempera-
ture, salt and extreme pH [16,17,18]. It is not surprising
that modern microorganisms have adopted a common
strategy to cope with various environmental stresses given
the natural history of the Earth (Box 1).
In extreme environments, such as the deep-sea with HHP,
cells suffer from an imbalance in oxidation and reduction,
insufficient energy supply and unstable macromolecules
[19]. The excessive reactive oxygen species (ROS) gen-
erated from the imbalance in oxidation and reduction can
cause cellular damage [20]. Anti-oxidation genes/proteins
are commonly found in piezophiles. Other genes involved
in HHP adaptation are responsible for maintaining the
energy supply and the native composition of proteins,
DNA and cell membranes. These genes (e.g., Hsp70,
RecA, Hsp60, F1F0 ATPases, Cct and UvrA) are also
involved in the adaptation to many other extremes. In
addition, piezophiles accumulate low-molecular-weight
osmolytes, mainly organic solutes, which are responsible
for extreme cold, heat, salinity and pH responses [18]
(Figure 2). Many of these solutes offer protective proper-
ties, such as cellular metabolic protection, by serving as
antioxidants that scavenge the free radicals and reactive
oxygen species generated under stress [21].
This common adaptation strategy allows microorganisms
to simultaneously adapt to HHP along with multiple other
stresses. When facing the multiple stresses, some common
genes/proteins were employed, such as Hsp70 and Clp.
These genes/proteins may also play a role in HHP adapta-
tion, and still need to be tested. HHP is not a strict
thermodynamic parameter, and some shallow species

Figure 2

Oxidation Salt
Glutamate, glycine, betaine, trehalose Glutamate, glycine, betaine, TMAO
Uv
Cc rA, es
tL DC P as
ex lp
A AT lp
Re Htp F0 2 C
Hsp70 RecA Hsp60 Cct
cN G H F1 at1,
0
Tat 1,2 Clp Hsp31 ion M sp p7 t T
Hsp30 Hsp104 Ino1 rr 10
Su 4 Hs Cc
lA Hsp70 Rec A Hsp60 Cct
Glutamate, TMAO, trehalose Hsp31 HtpG Hsp30 LexA
RecN uvrA,D ole1
Temperature Pressure Acid
Glutamate
F1F0 ATPases Hsp70 RecA Hs
Hsp60 Clp PAU OmpH opi3 p7
0
ole1 HtpG uvrA,D Cl
p Cc F1F
2 t T 0A
B-hydroxybutarate t1, at1 TP
Ta ,2 a
Clp ses

Trehalose, glutamate, glycine, betaine, TMAO Glutamate, glycine, betaine, TMAO

Osmosis Starvation

Membrane, DNA, Protein, Metabolism, Piezolyte

Current Opinion in Biotechnology

The relationship between adaptation strategies to high pressure and other extremes. The genes responsive to HHP were selected according
to [18], and if their analogs are responsive to other environmental stresses, for example, oxidative stress, their names were indicated between
pressure and oxidation. Different colors indicated the related functions of genes or proteins as illustrated below. The genes listed above
‘Temperature’ are the ones responsive to high temperature adaptation, whereas the ones listed below are the ones responsive to low temperature.

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160 Environmental biotechnology
(such as Escherichia coli) could tolerate high hydrostatic
pressure up to 50 MPa. HHP has a close relationship with
other stresses including nutrient limitation and extreme
pH/Tm/salt. For instance, upon shifting to HHP environ-
ments, cells may also face nutrient limitations. Under low
nutrient conditions, Psychromonas sp. CNPT3 cells in-
crease their ability to attach to glass surfaces with sub-
strates, particularly when under high pressure [22]. This
property enables deep-sea bacteria to attach to sediment
particles, the major food resource in the deep biosphere.
Another example is the nitrate reduction system of marine
Shewanella strains, which is a clear evolutionary adaptation
for survival at great water depths and allows microorgan-
isms to efficiently utilize the nitrate available in the local
environment [23]. Adaptation to multiple stresses will
provide scientific guidance for HHP cultivation and piezo-
phile application.
Sampling and cultivation of deep-sea
microorganisms
Since the end of 1940s and the time of ZoBell [24,25],
researchers have been aware that piezophiles are sensitive
to pressure shifts and that they may not survive transport
from the sea bottom to the surface. Parkes et al. proposed a
system for recovering and processing sediments under in
situ pressures, allowing experiments to be conducted in
the laboratory without depressurization [26]. These tar-
geted pressurized devices are designed to obtain and
manipulate samples with effective sealing, low distur-
bance and no contamination.
This HHP sampling equipment can be operated by
research vessels and submersibles or by direct fixation
to drilling platforms. The equipment is normally
launched with cameras and sensors to collect images
and data from the environment. A few pressure-retaining
water samplers and autoclave sediment cores have been
successfully deployed over the past decade [26]. Water
samplers are normally sealed with ball valves, whereas
sediment corers are sealed with flappy valves. In situ
filtration is occasionally applied to increase the biomass
concentration. Submersible-assisted sampling offers great
advantages, such as precise positioning, flexibility, and
mobility, and the manipulators associated with submer-
sibles can provide operational assistance (Box 2).
After samples arrive onboard, subsampling can be per-
formed by pushing the water or sediments into another
vessel under static pressure. Analysis and enrichment can
be performed either directly after subsampling or after the
subsamples are transferred to a laboratory. Subsampling
allows the samples to be used more efficiently. For sedi-
ment cores, subsamples from different layers can be stored
and cultivated independently without mixing [27]. The
subsample vessels should be specially designed, with
modification from laboratory-based pressurized incubation
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vessels, to be capable of surviving long transport distances
without depressurization.
Pressurized cultivation devices were invented and ap-
plied long before pressurized samplers. These devices
have been used to perform many types of laboratory
analyses and experiments, including prokaryotic enrich-
ment and isolation and measurement of bacterial activity
under HHP. Depending on the purpose of the experi-
ments, HHP cultivation can be performed in volumes of
less than one milliliter to tens of liters, either in batches or
continuously, even with gas as a substrate [15,28,29]. The
cultivation vessels are normally equipped with a pressure
gauge, thermal jacket, safety valve, stirring paddle and
occasionally environmental sensors and windows to mon-
itor the biochemical processes (Box 2).
Genetic engineering of piezophilic
microorganisms
The development of useful genetic tools has enabled
intensive investigation of HHP adaptation on the level of
molecular genetics. Genetic systems based on piezophilic
bacterial strains have been constructed, including psy-
chro-piezophiles such as Photobacterium profundum SS9
and Shewanella piezotolerans WP3 (Figure 1). For P. pro-
fundum SS9, an entire set of conjugal vectors for cloning,
expression and insertional mutagenesis has been devel-
oped [30]. Using these versatile vectors, gene disruption,
complementation and expression have been performed in
P. profundum SS9, resulting in a much better understand-
ing of the survival strategy under HHP [31–33]. For S.
piezotolerans WP3, a similar conjugation to that used for P.
profundum SS9 has been employed with a vector engi-
neered from the filamentous phage SW1 that is inducible
at the low temperatures employed [23,34–36]. Based on
this new vector, gene complementation and expression
have been performed in P. profundum SS9 and S. piezo-
tolerans WP3.
The most recognized genetic systems for piezophilic
Archaea have been developed in the hyperthermo-piezo-
philes Pyrococcus abyssi GE5 [37], P. yayanosii [38] and
Thermococcus barophilus MP [39]. Because most isolated
piezophilic archaea belong to the order Thermococcales, the
genetic systems for piezophilic archaea are often modified
from non-piezophilic Thermococcales, for example, Thermo-
coccus kodakaraensis KOD1. The establishment of a gene
disruption system for T. kodakaraensis KOD1 was a signifi-
cant breakthrough [40,41]. Moreover, overexpression of
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) re-
ductase was found to confer resistance to simvastatin,
making it possible to select for Thermococcales transformants
on nutrient-rich media under high temperatures [42,43]. A
gene disruption system for the deep-sea hyperthermophil-
ic and piezophilic archaeon P. yayanosii was developed
using simvastatin and 5-FOA as selection and counter-
selection markers, respectively [38]. Auxotrophic markers
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 HHP review Zhang et al. 161

Box 2 Sampling and cultivation devices of deep sea piezophiles.

Submersibles are normally equipped with a robotic arm, which can assist in location and the operation of devices underwater. This ability allows us
to perform complex, multiple step sampling processes. For example, a recently designed submersible based sediment sampler (a) was
constructed with a screw closing and two ball valves, which allows the retention of higher pressures than cores sealed with flappy valves and
provide a direct connection to HHP cultivation systems as incubation vessels (b) without depressurization. However, submersibles require relatively
low-weight devices and have limited sampling capacity. The weight problem can be partially solved by using an elevator to transport between the
ship and the seafloor. The recently developed in situ hydrothermal vent simulator can control various environmental parameters, such as gas
pressure, hydrostatic pressure, substrate composition, flow rate and temperature (the scheme was shown in b and the picture was shown in c).

(a) (c)

(b) PG
Gas
Inlet

Motor Stirring
Back
Sampling Pressure
Port Regulator
Gas Charging Pipet
Safety Pump Safety
Disk Disk
PG PG

Filter
Safety
Valve Sampling Safety
Port Valve

Heating Medium Effluent

Jacket Bottle Bottle
Heating
Conditioning Sampling Jacket
Vessel Port

Incubation
Pump Vessel
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162 Environmental biotechnology
were used for genetic manipulation, for example, through
tryptophan auxotrophy, uracil auxotrophy and arginine
auxotrophy, because of the harsh environments of
hyperthermo-piezophiles. The shuttle vector pYS2 con-
tains the pyrE gene of Sulfolobus acidocaldarius, which was
engineered by fusing to the endogenous plasmid pGT5
and the bacterial vector pLitmus38 and was used in
conjunction with a PEG spheroplast method to transform
the pyrE mutant of P. abyssi GE5 while selecting for uracil
prototrophy [37].
With these available genetic engineering systems, the
hypothesis of HHP adaptation could be tested. For
example, the redox sensor could be introduced into cells
and used to detect the physiologic responses under HHP
in vivo. The restriction-modification system can be
knocked out/in, piezolytes can be overexpressed in vivo,
and the capability of stress tolerance could then be tested.
Possible future applications
Deep-sea environments have been heralded as a new
target environment for the search for novel and valuable
bioactive compounds [44,45]. For example, both culture-
independent [46] and culture-dependent [47]
approaches have been successfully used to detect actino-
mycetes, well-known producers of bioactive secondary
metabolites, in deep-sea sediments. In addition, many
psychrophilic and piezophilic bacteria contain omega-3-
polyunsaturated fatty acids (PUFAs) associated with their
membrane lipids, particularly eicosapentaenoic acid
(EPA; 20:5n3) and docosahexaenoic acid (DHA;
22:6n3), to counter the membrane ordering effects of
decreasing temperature or increasing pressure [48]. Ome-
ga-3 PUFAs are essential fatty acids and precursors to
hormones and hormone-like molecules in many animals.
Moreover, pressure-inducible osmolytes, termed piezo-
lytes, could function by facilitating the appropriate struc-
ture and function of proteins at high pressure in a manner
analogous to that of the high temperature stabilizing
osmolytes present in hyperthermophiles [49]. Addition-
ally, Deep VentR DNA Polymerase derived from a deep-
sea hydrothermal vent Pyrococcus species is available from
New England Biolabs. Given that these enzymes have
evolved to function at low activation volume change as
well as high temperature, their enzymatic properties are
likely to be different from shallower enzyme sources, and
as a result, potentially useful. Piezophiles or their pro-
ducts could be introduced with or without modification
into various high-pressure processes. For example, obli-
gate piezophiles could represent the ultimate biological
containment system for the production of lethal toxins,
virus particles and virulence factors, without posing a
threat to the surrounding environments at ambient atmo-
spheric pressures. As intriguing as this idea may be, it is
not yet practical due to the lack of genetic technologies
for microorganisms that grow and survive at only elevated
pressures. Another practical application for piezophiles
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lies in the field of high pressure processing of foods and
beverages. High-pressure food pasteurization, also re-
ferred to as pascalization, as well as high pressure food
processing, are the largest activities in high-pressure
biotechnology today and are still growing. While much
of the focus of this industry is on the inactivation of
pathogens and enzymes, in some cases, obtaining advan-
tageous enzymes that display resistance to high-pressure
inactivation, have selective activity at high pressure, or
possess broad pressure ranges of enzyme activity would be
a desirable outcome. For example, proteolysis is a neces-
sary component of cheese ripening. This activity could be
modified during pascalization, and as a result, genetic
changes would be introduced into the starter bacteria
present in cheese, such as lactic acid bacteria [50]. We
suggest that increased dialog between those working on
piezophiles, enzyme functions at high pressure and high-
pressure food and beverage processing holds many possi-
bilities for the innovation of new products.
Perspectives
At least three gaps in the understanding of HHP adapta-
tion remain. First, of the large and small ribosomal sub-
units that have been recovered, less than 1% have been
verified as members of the piezophilic Gammaproteobac-
terial families Colwelliaceae, Moritellaceae, Psychromo-
nadaceae and Vibrionaceae. Members of the phyla
Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi,
Deferribacteres and Planctomycetes constitute the major
remaining rRNA classifications outside of the Proteobac-
teria. Because most of our knowledge of HHP adaptation
has been generated based on isolated strains, it is impor-
tant to determine whether most uncultivated piezophiles
follow the same rule. Second, all of the known piezophiles
have been isolated using rich medium containing approx-
imately 1% peptone and yeast extract, for example,
2216E (Zobell 2216E medium) and TRM (Thermococ-
cales Rich Medium). This isolation method only applies
to R-strategist piezophiles, which have high growth rates
and short generation times. In contrast with R-strategist
piezophiles, K-strategist piezophiles have longer genera-
tion times, lower growth rates and smaller populations.
Can we somehow succeed at isolating K-strategist piezo-
philes using another approach, such as limiting the nutri-
ent supply? Moreover, are the K-strategist piezophiles
using the same adaptation to cope with HHP? Third,
although both obligate piezophilic bacteria and archaea
have been isolated, all of the genetic systems to date have
been based on facultative piezophilic and piezotolerant
strains. Thus, it is important to determine whether the
genes and pathways responsible for the obligate piezo-
philic characteristics of these organisms are dispensable or
central to high-pressure adaptation.
As R.E. Marquies anticipated in 1982, obligate piezo-
philic bacteria (no archaea at that time) and piezophilic
mutant strains of common bacteria will be an asset to
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future research. P. yayanosii CH1, isolated from the hy-
drothermal vent ‘Ashadze’ (4100 m depth), was the first
strict piezophilic anaerobic hyperthermophilic archaeon.
Recently, a facultative-piezophilic derivative strain with
pressure-independent growth has been obtained using
random foreign DNA fragment transformation [38]. In
parallel, a similar facultative piezophilic derivative strain
has also been isolated through stepwise reductions of
temperature and pressure (PM Oger, personal commu-
nication). Omics and physiological comparison of these
spontaneous mutants may provide a long-awaited an-
swer to the question of how absolute HHP adaptation
occurred.
Moreover, newly designed equipment, especially flow-
through cultivation systems, can mimic multiple envi-
ronmental factors, including oligotrophic processes, to
help isolate other types of obligate piezophiles, includ-
ing K-strategists. In the future, novel sampling devices
can be designed that add certain reagent in situ, such as
antioxidants, to reduce the sensitivity of (obligate)
piezophiles to ambient pressure. A better understand-
ing of HHP microorganisms and their adaptation strat-
egies will certainly facilitate the development of novel
biotechnologies.
Acknowledgements
This work was supported by the National Basic Research Program of China
(2014CB441500), National Science Foundation of China (31290232),
National Science Foundation of China (41102211), National High-Tech
Program of China (2012AA092103), State Key Laboratory of Microbial
Metabolism (Shanghai Jiao Tong University), SKL Ocean Engineering of
China (GKZD010061).
References and recommended reading
Papers of particular interest, published within the period of review,
have been highlighted as:
 of special interest
 of outstanding interest
1. Tyler P: Ecosystems of the Deep Ocean. Elsevier Science Ltd.;
2003.
2. Zobell CE, Johnson FH: The influence of hydrostatic pressure
on the growth and viability of terrestrial and marine bacteria.
J Bacteriol 1949, 57:179-189.
3. Yayanos AA: Microbiology to 10,500 meters in the deep sea.
Annu Rev Microbiol 1995, 49:777-805.
4. Fang JS, Zhang L, Bazylinski DA: Deep-sea piezosphere and
piezophiles: geomicrobiology and biogeochemistry. Trends
Microbiol 2010, 18:413-422.
5. Kato C: Distribution of piezophiles.Extremophiles Handbook
Tokyo: Springer Verlag; 2011, 643-655.
6. Kim S-J, Kato C: Sampling, isolation, cultivation, and
characterization of piezophilic microbes.Handbook of
Hydrocarbon and Lipid Microbiology Berlin Heidelberg:
Springer-Verlag; 2010, 3869-3881.
7. Kato C, Nogi Y, Arakawa S: Isolation, cultivation, and diversity
of deep-sea piezophiles. High-pressure Microbiology.
Washington, DC: ASM Press; 2008, 203-217.
8. Yayanos AA, Dietz AS, Van Boxtel R: Obligately barophilic
bacterium from the Mariana trench. Proc Natl Acad Sci USA
1981, 78:5212-5215.
www.sciencedirect.com
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For personal use only. No other uses without permission. Copyright ©2017. Elsevier Inc. All rights reserved.
HHP review Zhang et al. 163
9. Yayanos AA: Piezophiles. Chichester: John Wiley & Sons Ltd.;
2008 http://dx.doi.org/10.1002/9780470015902.a0000341.pub2:.
http://www.els.net.
10. Birrien J-L, Zeng X, Jebbar M, Cambon-Bonavita M-A,
Quérellou J, Oger P, Bienvenu N, Xiao X, Prieur D: Pyrococcus
yayanosii sp. nov., an obligate piezophilic hyperthermophilic
archaeon isolated from a deep-sea hydrothermal vent.
Int J Syst Evol Microbiol 2011, 61:2827-2881.
11. Zeng X, Birrien JL, Fouquet Y, Cherkashov G, Jebbar M,
Querellou J, Oger P, Cambon-Bonavita MA, Xiao X, Prieur D:
Pyrococcus CH1, an obligate piezophilic hyperthermophile:
extending the upper pressure-temperature limits for life. ISME
J 2009, 3:873-876.
12. Eloe EA, Fadrosh DW, Novotny M, Allen LZ, Kim M, Lombardo MJ,
Yee-Greenbaum J, Yooseph S, Allen EE, Lasken R et al.: Going
deeper: metagenome of a hadopelagic microbial community.
PLoS ONE 2011, 6:e20388.
13. Lauro FM, Bartlett DH: Prokaryotic lifestyles in deep sea
habitats. Extremophiles 2008, 12:15-25.
14. Yooseph S, Nealson KH, Rusch DB, McCrow JP, Dupont CL,
Kim M, Johnson J, Montgomery R, Ferriera S, Beeson K et al.:
Genomic and functional adaptation in surface ocean
planktonic prokaryotes. Nature 2010, 468:60-66.
15. Bartlett DH, Kerman I: Contributions of large-scale DNA
sequencing efforts to the understanding of low temperature
piezophiles.Extremophiles Handbook Tokyo: Springer Verlag;
2011, 703-718.
16. Oger PM, Jebbar M: The many ways of coping with pressure.
Res Microbiol 2010, 161:799-809.
17. Harrison JP, Gheeraert N, Tsigelnitskiy D, Cockell CS: The limits
 for life under multiple extremes. Trends Microbiol 2013, 21:204-
212.
Summerizes the combine effect of HHP with T, pH, or salinity on microbial
growth rates based on the published data from 67 prokaryotic strains.
18. Ambily Nath IV, Loka Bharathi PA: Diversity in transcripts and
translational pattern of stress proteins in marine
extremophiles. Extremophiles 2011, 15:129-153.
19. Xiao X, Zhang Y: Life in extreme environments: approaches
 to study life-environment co-evolutionary strategies. Sci China
Earth Sci 2014, 57:869-877.
Illustrates the general adaptation stratergies of microorganims to HHP
and other types of environmental stress.
20. Poljsak B, Šuput D, Milisav I: Achieving the balance between
ROS and antioxidants: when to use the synthetic antioxidants.
Oxid Med Cell Longev 2013:2013.
21. Yancey PH: Organic osmolytes as compatible, metabolic and
counteracting cytoprotectants in high osmolarity and other
stresses. J Exp Biol 2005, 208:2819-2830.
22. Rice SA, Oliver JD: Starvation response of the marine barophile
CNPT-3. Appl Environ Microbiol 1992, 58:2432-2437.
23. Chen Y, Wang F, Xu J, Mehmood MA, Xiao X: Physiological and
evolutionary studies of NAP systems in Shewanella
piezotolerans WP3. ISME J 2011, 5:843-855.
24. Demazeau G, Rivalain N: High hydrostatic pressure and biology:
a brief history. Appl Microbiol Biotechnol 2011, 89:1305-1314.
25. Michiels C, Bartlett DH, Aertsen A: High-pressure Microbiology.
Washington (D.C): ASM Press; 2008.
26. Parkes RJ, Martin D, Amann H, Anders E, Holland M,
Schultheiss PJ, Wang X, Dotchev K: Technology for high-
pressure sampling and analysis of deep-sea sediments,
associated gas hydrates, and deep biosphere processes. In
Natural Gas Hydrates—Energy Resource Potential and associated
Geologic Hazards. Edited by Collett T, Johnson A, Knapp C,
Boswell R. AAPG/NETL/AAPG Foundation/AAPG EMD; 2010:145.
27. Parkes RJ, Sellek G, Webster G, Martin D, Anders E,
Weightman AJ, Sass H: Culturable prokaryotic diversity of
deep, gas hydrate sediments: first use of a continuous high-
pressure, anaerobic, enrichment and isolation system for
Current Opinion in Biotechnology 2015, 33:157–164
164 Environmental biotechnology
subseafloor sediments (DeepIsoBUG). Environ Microbiol 2009,
11:3140-3153.
28. Zhang Y, Arends JBA, Van de Wiele T, Boon N: Bioreactor
technology in marine microbiology: from design to future
application. Biotechnol Adv 2011, 29:312-321.
29. Kato C: Cultivation methods for piezophiles.Extremophiles
Handbook Tokyo: Springer Verlag; 2011, 719-726.
30. Lauro FM, Eloe EA, Liverani N, Bertoloni G, Bartlett DH: Conjugal
vectors for cloning, expression, and insertional mutagenesis
in gram-negative bacteria. BioTechniques 2005, 38:708-712.
31. Lauro FM, Tran K, Vezzi A, Vitulo N, Valle G, Bartlett DH:
Large-scale transposon mutagenesis of Photobacterium
profundum SS9 reveals new genetic loci important for growth
at low temperature and high pressure. J Bacteriol 2008,
190:1699-1709.
32. Eloe EA, Lauro FM, Vogel RF, Bartlett DH: The deep-sea
bacterium Photobacterium profundum SS9 utilizes separate
flagellar systems for swimming and swarming under high-
pressure conditions. Appl Environ Microbiol 2008, 74:6298-6305.
33. Simonato F, Campanaro S, Lauro FM, Vezzi A, D’Angelo M,
Vitulo N, Valle G, Bartlett DH: Piezophilic adaptation: a genomic
point of view. J Biotechnol 2006, 126:11-25.
34. Wang F, Li Q, Xiao X: A novel filamentous phage from the deep-
sea bacterium Shewanella piezotolerans WP3 is induced at
low temperature. J Bacteriol 2007, 189:7151-7153.
35. Wang F, Xiao X, Ou HY, Gai Y: Role and regulation of fatty acid
biosynthesis in the response of Shewanella piezotolerans
WP3 to different temperatures and pressures. J Bacteriol 2009,
191:2574-2584.
36. Jian H, Xiao X, Wang F: Role of filamentous phage SW1 in
regulating the lateral flagella of Shewanella piezotolerans
strain WP3 at low temperatures. Appl Environ Microbiol 2013,
79:7101-7109.
37. Lucas S, Toffin L, Zivanovic Y, Charlier D, Moussard H, Forterre P,
Prieur D, Erauso G: Construction of a shuttle vector for, and
spheroplast transformation of, the hyperthermophilic
archaeon Pyrococcus abyssi. Appl Environ Microbiol 2002,
68:5528-5536.
38. Li X, Fu L, Li Z, Ma X, Xiao X, Xu J: Genetic tools for the
piezophilic hyperthermophilic archaeon Pyrococcus
yayanosii. Extremophiles 2015, 19:59-67.
39. Thiel A, Michoud G, Moalic Y, Flament D, Jebbar M: Genetic
manipulations of the hyperthermophilic piezophilic archaeon
Thermococcus barophilus. Appl Environ Microbiol 2014,
80:2299-2306.
40. Sato T, Fukui T, Atomi H, Imanaka T: Targeted gene disruption
 by homologous recombination in the hyperthermophilic
archaeon Thermococcus kodakaraensis KOD1. J Bacteriol
2003, 185:210-220.
Demonstrates the genetic manipulations on piezophilic archaea appling
the most recently developed genetic tools.
Current Opinion in Biotechnology 2015, 33:157–164
Downloaded for FK UMI 3 Makassar (fkumimks3@gmail.com) at ClinicalKey Global Flex Package Trial from ClinicalKey.com by Elsevier on November 07, 2017.
For personal use only. No other uses without permission. Copyright ©2017. Elsevier Inc. All rights reserved.
41. Sato T, Fukui T, Atomi H, Imanaka T: Improved and versatile
transformation system allowing multiple genetic
manipulations of the hyperthermophilic archaeon
Thermococcus kodakaraensis. Appl Environ Microbiol 2005,
71:3889-3899.
42. Matsumi R, Manabe K, Fukui T, Atomi H, Imanaka T: Disruption
of a sugar transporter gene cluster in a hyperthermophilic
archaeon using a host-marker system based on antibiotic
resistance. J Bacteriol 2007, 189:2683-2691.
43. Waege I, Schmid G, Thumann S, Thomm M, Hausner W: Shuttle
vector-based transformation system for Pyrococcus furiosus.
Appl Environ Microbiol 2010, 76:3308-3313.
44. Bhatnagar I, Kim SK: Immense essence of excellence: marine
microbial bioactive compounds. Mar Drugs 2010, 8:2673-2701.
45. Hopwood DA: Therapeutic treasures from the deep. Nat Chem
Biol 2007, 3:457-458.
46. Prieto-Davo A, Villarreal-Gomez LJ, Forschner-Dancause S,
 Bull AT, Stach JE, Smith DC, Rowley DC, Jensen PR: Targeted
search for actinomycetes from nearshore and deep-sea
marine sediments. FEMS Microbiol Ecol 2013, 84:510-518.
Outlook on the potential of deep-marine actinomycetes.
47. Subramani R, Aalbersberg W: Culturable rare Actinomycetes:
diversity, isolation and marine natural product discovery.
Appl Microbiol Biotechnol 2013, 97:9291-9321.
48. Usui K, Hiraki T, Kawamoto J, Kurihara T, Nogi Y, Kato C, Abe F:
Eicosapentaenoic acid plays a role in stabilizing dynamic
membrane structure in the deep-sea piezophile Shewanella
violacea: a study employing high-pressure time-resolved
fluorescence anisotropy measurement. Biochim Biophys Acta
2012, 1818:574-583.
49. Neves C, da Costa MS, Santos H: Compatible solutes of the
hyperthermophile Palaeococcus ferrophilus: osmoadaptation
and thermoadaptation in the order thermococcales. Appl
Environ Microbiol 2005, 71:8091-8098.
50. Katsaros GI, Giannoglou MN, Taoukis PS: Kinetic study of the
combined effect of high hydrostatic pressure and temperature
on the activity of Lactobacillus delbrueckii ssp. bulgaricus
aminopeptidases. J Food Sci 2009, 74:E219-E225.
51. Knauth LP: Temperature and salinity history of the
Precambrian ocean: implications for the course of microbial
evolution. Palaeogeogr Palaeoclimatol Palaeoecol 2005,
219:53-69.
52. Bekker A, Holland HD, Wang PL, Rumble D, Stein HJ, Hannah JL,
Coetzee LL, Beukes NJ: Dating the rise of atmospheric oxygen.
Nature 2004, 427:117-120.
53. Partin CA, Bekker A, Planavsky NJ, Scott CT, Gill BC, Li C,
Podkovyrov V, Maslov A, Konhauser KO, Lalonde SV et al.: Large-
scale fluctuations in Precambrian atmospheric and oceanic
oxygen levels from the record of U in shales. Earth Planet Sci
Lett 2013, 369:284-293.
www.sciencedirect.com