Piezophilic And Non Piezophilic Growth Conditions

Published Date: 02 Nov 2017

Introduction:

The ocean, with an average depth of 3800m, and pressure of 38 MPa (0.1 megapascals = 1 bar=0.9869 atmospheres=1.0197 kgfcm-2; to avoid confusion megapascals [MPa] is used throughout), comprises ~70% of the biosphere. At the bottom of the Mariana Trench, the deepest known site in the ocean, the pressure is 110 MPa. Despite the fact that a major proportion of the Earth’s biosphere is a high-pressure environment, much less is known about deep-sea Piezophilic (previously barophilic) microorganism than soil microorganisms. The temperature in the deep-sea is typically within the range of 1-30C. The discovery of hydrothermal vents in the sea floor has revealed habitats where high pressure and high temperature conditions exist. Thus, marine organisms inhabit environments where they might be exposed to a temperature range of 1-3000C and pressures that range from 0.1-110 MPa. Deep-sea organisms have attracted the attention of marine biologists who wish to understand the survival strategies employed under such extreme conditions. The presence of bacteria in deep ocean waters and sediments was one of the first discoveries in marine microbiology (Certes, 1884) and subsequent studies suggest that they may form a major component of particulate organic material in aphotic oceanic waters (Cho and Azam, 1988). Our knowledge concerning the physical conditions under which the microbes can grow has increased significantly in recent years. In most of the deep sea, microorganisms grow at 2 to 30C and hundreds of bars of hydrostatic pressure. At nearly 11,000m, the challenger Deep is the deepest known oceanic site, and the microbes that are active there must be able to function at pressures greater than 100 MPa.

Review of Literature:

The deep-sea is regarded as an extreme environment with conditions of high hydrostatic pressure [up to 110 megapascals (MPa)], and predominantly low temperature (1-20C). It is accepted that deep-sea microbiology as a definable field did not exist before the middle of this century and little attention was paid to this field except for the efforts of Certes and Portier ( Jannasch and Taylor, 1984). Certes, during the Travaillier and Talisman Expeditions (1882-1883), examined sediment and water collected from depths to 5,000m and found bacteria in almost every sample. In 1949, ZoBell and Johnson (1949) started work on the effect of hydrostatic pressure on microbial activities. The term "barophilic" was first used, defined as optimal growth at a pressure higher than 0.1MPa or a requirement for increased pressure for growth.

Dark Ocean:

The dark ocean can be considered any habitat existing below the photic zone of the ocean. Other than permanent darkness, another common unifying feature of the dark ocean is relatively high pressures (pressure increases by ~ 1 atm with every 10 m of water depth). Habitats in the dark ocean span a wide range of temperatures, from relatively cold (some below 0C) deep water masses to high-temperature hydrothermal vents (up to 400C in some places). By volume, low temperature and high pressure dominate habitable dark ocean environments. Although such conditions are often referred to as "extreme," considering their ubiquity in the environment, these conditions are actually quite average on a global scale.

The majority of the Earth’s habitable environments are physically located in environments that do not receive sunlight. Indeed, the largest potential habitats on Earth are located in the ocean, which covers approximately 70% of the Earth’s surface. The ocean’s average depth is 4,000 m and reaches as deep as 11,000 m in the Marianas Trench. Considering that other habitats exist beneath the ocean water column, such as marine sediments, oceanic crust, and hydrothermal vents, these dark ocean environments together comprise the largest collection of habitats by volume that life in particular, microbial life can occupy on Earth. Accordingly, research is focussing on the isolation and cultivation of microorganisms from extreme environments. Several environments can be regarded as extreme; these are summarized in table 1.

Table 1. Definition of extreme condition for specific environmental variables.

Environmental variable extreme conditions

pH <pH 3 or pH 9

temperature <100C or >700C

Salinity >15% (w/v)NaCl

Organic solvents (eg: toluene > 1 % (w/v)

Heavy metals (e.g: Hg and Cd >M)

Pressure >50 MPa (50 atm)

Microbial Distribution and Diversity:

Our knowledge of microbial processes in the dark ocean has increased enormously in recent decades, owing in part to the exciting discoveries of hydrothermal vents, cold seeps, and whale falls at the bottom of the ocean in the late 1970s and 1980s. Studies that try to decipher the activity of microorganisms in the dark ocean, where we cannot easily observe them, continually yield paradigm-shifting discoveries, fundamentally changing our understanding of the role of the dark ocean in the global Earth system and its biogeochemical cycles. As one example, the discovery of the existence and predominance of psychrophilic and mesophilic organisms below the photic zone in the worlds ocean has radically changed our understanding of the distribution of archaea on Earth and raised questions about the function of the organisms in this global habitat. Another revolutionary discovery emerged from research of deeply buried sediments of the dark ocean, where active microorganisms are now known to persist in sediments hundreds of meters below the ocean floor that are millions of years old. These discoveries prompted a recent wave of studies to understand the extent, function, and importance of a deep sedimentary biosphere in the dark ocean.

Microbes are the principle custodians of the environment, balancing and maintaining earth’s global biogeochemical cycles. Although researchers have recognized for many years the importance of studying the dark ocean, our understanding of this realm has lagged behind that of its sunlit counterparts in the terrestrial and marine realms due to the difficulty in accessing it, as studies in the dark ocean are both technically challenging and expensive.

Microbiologists studying life in the dark ocean and the potential for life on other planets are developing key unifying reference frames concerning "habitability" of different environments. The study of microorganisms isolated from deep-sea habitats is providing insight into the ecology and evolution of life in high pressure environments. Elevated hydrostatic pressure is an important thermodynamic parameter that has greatly influenced the physiological and biochemical adaptations of marine organisms inhabiting different depths. At the molecular level, the responses of microorganisms that are not high-pressure adapted to elevated pressures have revealed fundamental differences in cellular metabolism and regulatory processes as compared to microorganisms that specifically thrive under elevated pressures. Analysis of nonpiezophilic microorganisms at elevated pressures hint at pressure-sensitive cellular phenomena which Piezophilic microorganisms must modify for high-pressure adaptation.

Deep-Sea Habitats:

The deep sea can broadly be characterized by the presence of high hydrostatic pressures (upto 1100 atm or 110 megapascal (MPa)), generally low temperatures ~20C except in regions of hydrothermal activity (up to 3800C), the absence of light, and general oligotrophy. As such, the deep sea can be regarded as an extreme environment. Throughout the spectrum of physicochemical parameters encountered in deep sea, microbial life exists. The nature of deep-sea habitats is determined by numerous factors including input of surface derived nutrients, geochemical, and geothermal influences, and physical oceanographic and hydrological regimes in addition to the pressure and the temperature. These factors in turn govern the local community structure and biodiversity of the habitat. Microbiologically relevant high pressure environments encountered on Earth are listed in Table 1.

Environment

Approximate Pressure

Deep-sea water column/surface sediments

112 MPa

Deep-sea invertebrates

108 MPa

Deep-sea fish

63 MPa

Deep-sea brines

15 MPa

Hydrothermal vents

41 MPa

Deep marine sediments

14 MPa

Deep basaltic rock

67 MPa

Deep granite rock

55 MPa

Deep oil reservoirs

31 MPa

Table 1. High-pressure microbiological environments on Earth and their documented approximate upper pressures

Piezophiles:

In deep sea realm where the microbes form the predominant biotic community, the distribution and survival of organisms is controlled by the crucial factor, pressure (Whitman et al., 1998; somero, 1990; Yayanos, 1986). Deep sea habitat is characterized by a lower temperature limit of -7.5C (Bedford, 1933), and the upper temperature limit is reported to be 113C (Blochl et al., 1997). A microorganism can be assigned to one of several groups depending on its response to elevated pressure. Those which can grow at pressures encountered in the deep-ocean may be defined as piezotolerant if they grow optimally at atmospheric pressure or Piezophilic(barophilic) if they require a high pressure for optimal growth ( Yayanos, 1998; Fang et al., 2010). An organism which grows optimally at atmospheric pressure but whose growth is inhibited by moderate pressure may be referred to as piezosensitive. Piezophiles are microorganisms that possess optimal growth rates at pressures above atmospheric pressure. These microbes inhabiting the deep sea can grow at 20C to 3C and hundreds of bars of hydrostatic pressure and those living in challenger Deep, which is the deepest known oceanic site, must be adapted to survive at pressures>100 megapascals (MPa) (1bar=0.1MPa. presently the term "Piezophilic" which was previously referred to as barophile, is used to describe those microorganisms with optimal growth at pressures>0.1Mpa. Piezotolerant microorganisms are capable of growth at high pressure, as well as at atmospheric pressure, but can be distinguished from Piezophiles because they do not have optimal growth rates at pressures above 1 atmosphere. Piezotolerant microbes can also be distinguished from piezosensitive microorganisms (whose growth is sensitive to elevated pressure) because they can grow at 50 MPa at a rate that is above 30% of their growth rate at atmospheric pressure, as long as they have otherwise optimal growth conditions.

The field of piezomicrobiology was born more than 100 years ago (Simanato et al., 2006), but is still in an infant stage because of the limited number of scientists and labs involved in the field most probably due to the lack of specialized and expensive collection vehicles. Many basic properties of Piezophiles that enable their survival at extreme pressures remain to be elucidated (Kato and Bartlett, 1997). The major challenges in piezophysiology are to discover whether the physiological responses of living cells are relevant to their growth and to identify the critical factors in cell viability and lethality under high pressure (Abr, 2007).

There is growing interest in understanding microbes and potential applications of them in extreme environments that have significant impacts on them (Simanto et al., 2006; Bartlett, 2001; Abe and Horikoshi, 2001; Abe et al., 1999; Yayanos, 1995). The biomedical applications of Piezophiles are wide ranging. Proteins extracted from obligate Piezophiles such as Photobacterium profundum SS9, Shewanella violacea DSS12, and Pyrococcus abyssi are adapted to work both at high pressures and low temperatures.

The major genera of cultivated barophiles include the genera Shewanella, Photobacterium, Colwellia, and Moritella, as well as a new unidentified group, and all of these organisms can be classified as copiotrophs. They are able to grow under very nutrient-limited conditions because they have a high specific affinity for substrates and the ability to use the limiting substrates. To date, none of these organisms have been characterized as barophilic. The deep sea is generally described as an oligotrophic environment that contains 0.03 to 0.2mM dissolved organic carbon. Limited work has been done on the growth responses of barophiles to reduced substrate levels, and in the studies that have been done the workers have used batch culture approaches. If the deep-sea barophiles that have been isolated and studied to date are obligately copiotrophic with respect to carbon sources, then there in situ growth activities may well vary with their level of nutrition.

Characteristics of Piezophilic Organisms:

In 1949, Zobell and Johnson started work on the effect of hydrostatic pressure on microbial activities. The term "barophilic" was first used by them, defined as optimal growth at a pressure higher than 0.1 MPa or a requirement for increased pressure for growth. Zobell and Morita obtained the first evidence of Piezophilic growth in mixed microbial cultures recovered from the deep-sea. The first isolate of pressure-adapted bacteria was reported by Yayanos et al. in 1970 and subsequently, many psychrophilic Piezophiles with various optimal growth pressures have been isolated and characterized physiologically and genetically. Recently, the term "Piezophilic" was proposed to replace "barophilic" as the prefixes "baro" and "piezo," derived from the Greek meaning "weight" and "pressure," respectively. Thus, the word "Piezophilic" may be more suitable than "barophilic" to describe bacteria that grow better at high pressure than at atmospheric pressure. The term "Piezophilic" bacteria" meaning high-pressure-loving bacteria. The history of piezophile studies is shown in Table 1.

1949

Definition of Piezophiles (barophiles)

1950~

Piezophiles isolation started and study of protein synthesis, cell division etc.

1979

1st Isolation of Piezophiles

1981

Isolation of obligatory piezophiles

1985

Piezophilic bacteria Shewanella benthica was defined

1988

Piezophilic bacteria Colwellia hadaliensis was defined

1989

Pressure-regulated outer membrane protein

1995

Analysis of pressure-regulated genes

1998

Piezophiles, Photobacterium profundum, Shewanella violacea, and Moritella japonica were defined

1999

Extremely Piezophiles Moritella yayanosii was defined

2000

Analysis of the pressure-regulated transcription mechanisms

2002

Obligatory Piezophiles Psychromonas kaikoae was defined

2002~

The study is in progress.

In addition to deep-sea environments, high pressure can be considered an important environment parameter within other habitats. For example, Lake Baikal in Siberia is the deepest surface exposed freshwater lake in the world, possessing a maximal depth of 1600m. Recent evidence suggests that Lake Vostok, a large freshwater lake located 3 to 4 km beneath the East Antarctic Ice Sheet contains bacteria in relatively high concentrations. Microbes existing in these habitats could also be adapted for optimal growth and survival under elevated pressure conditions.

Hydrostatic pressure is unique to the oceans. Hydrostatic pressure increases approximately 1 atm or 0.103 MPa (1 atm = 1.013bar = 0.1 MPa = 1.013 x 102 kPa) for every 10 m depth, compared with 2.26 atm per meter for lithostatic pressure. The deep sea is generally defined as water depths of 1000 m and greater. Thus, the deep sea covers 88% of the area and occupies 75% of the total volume of the global ocean. Therefore, the deep-sea biosphere (piezosphere), represents the largest biotope of the earth.

Investigation of life in a high pressure environment:

It has been suggested that life may have originated in the deep sea some 3.5 to 4 billion years ago, where it was protected from the damaging effects of ultra violet light. The deep sea is a particularly high pressure environment, and hydrostatic pressure would have been very important stimuli for early stages of life. Recently, scientists have proposed that life might have originated in deep sea hydrothermal vents, and thus it seems possible that high pressure-adapted, mechanisms of gene expression could represent a feature present during the early forms of life. Recently, it has been reported that the primary chemical reactions involved in polymerization of organic materials (i.e., amino acids) could have occurred in such an environment. Thus, the study of deep-sea microorganisms may not only enhance our understanding of particular adaptations to abyssal and hadal ocean realms, but may also provide valuable insights into the origin and evolution of all life.

Pressure environment:

Pressure is an environmental parameter that varies only between narrow limits and thus has little or no influence in most commonly studied microbial niche. However, in some specific niches and situations, the life and death of microorganisms are strongly affected by pressure. This is the case for Piezophilic and piezotolerant microorganisms (respectively requiring or tolerating high pressure during growth) living in the deep sea and the deep subsurface and also for nonpiezophiles that are subject to pascalization, an emerging process for preserving foods by treatment with ultrahigh pressure (100 to 1000 MPa). Although pressure, like temperature, is a thermodynamically well-known physical parameter, the effects of high pressure on microorganism remain poorly characterized, unlike those of heat. Some effects of pressure on biomolecules and biological systems that have been well studied in vitro and explained on the basis of thermodynamic principles are protein denaturation and phase transition in membranes. Therefore, most pressure effects on microorganisms observed in vivo, such as inhibition of key enzymes and processes and disruption of cellular structures and membranes.

Microorganisms that are adapted to normal atmospheric pressure (0.1 MPa), can often grow at pressures up to a few tens of megapascals, but only at a strongly reduced rate. The deep sea, on the other hand, with pressures ranging from 30 up to 100 MPa, constitutes a reservoir of Piezophilic and piezotolerant microorganisms, which have become a model for studying piezophysiology and to gain insight into cellular adaption stratergies for coping with high-pressure stress.

Traditional Handling of Piezophiles

In 1957, Zobell and Morita were first considering how to handle Piezophilic microorganisms living under the deep-sea floor. They developed a titanium pressure vessel that could produce a pressure of up to 100 MPa for the handling and cultivation of such microbes (Zobell and Morita, 1957). They attempted several times to isolate only piezotolerant deep-sea microbes, which showed better growth under atmospheric pressure conditions but also grew at high pressure.one reason for their inability to usolate pezophiles was that such extremophiles can be sensitive to drastic changes in pressure and temperature, and it may be very difficult to maintain the microbes at atmospheric pressure. However, Zobell and Morita made a great contribution to the handling of microbes in high-pressure microbiology, and many researchers began to study biological physiology under pressure conditions.

In 1979, Yayanos and co-workers succeded in isolating Piezophilic microorganisms from amphipods recovered from a depth of 5782m in the Philippine Trench using pressure-retaining traps. This was the first report on the isolation of a peizophilic microbe (Yayanos et al., 1979).