Discovering the roles of subsurface microorganisms: Progress and future of deep biosphere investigation

The discovery of the marine “deep biosphere”—microorganisms living deep below the seafloor—is one of the most significant and exciting discoveries since the ocean drilling program began more than 40 years ago. Study of the deep biosphere has become a research frontier and a hot spot both for geological and biological sciences. Here, we introduce the history of the discovery of the deep biosphere, and then we describe the types of environments for life below the seafloor, the energy sources for the living creatures, the diversity of organisms within the deep biosphere, and the new tools and technologies used in this research field. We will highlight several recently completed Integrated Ocean Drilling Program Expeditions, which targeted the subseafloor deep biosphere within the crust and sediments. Finally, future research directions and challenges of deep biosphere investigation towards uncovering the roles of subsurface microorganisms will be briefly addressed.     

The habitat and nature of early life

Earth is over 4,500 million years old. Massive bombardment of the planet took place for the first 500-700 million years, and the largest impacts would have been capable of sterilizing the planet. Probably until 4,000 million years ago or later, occasional impacts might have heated the ocean over 100 degrees C. Life on Earth dates from before about 3,800 million years ago, and is likely to have gone through one or more hot-ocean 'bottlenecks'. Only hyperthermophiles (organisms optimally living in water at 80-110 degrees C) would have survived. It is possible that early life diversified near hydrothermal vents, but hypotheses that life first occupied other pre-bottleneck habitats are tenable (including transfer from Mars on ejecta from impacts there). Early hyperthermophile life, probably near hydrothermal systems, may have been non-photosynthetic, and many housekeeping proteins and biochemical processes may have an original hydrothermal heritage. The development of anoxygenic and then oxygenic photosynthesis would have allowed life to escape the hydrothermal setting. By about 3,500 million years ago, most of the principal biochemical pathways that sustain the modern biosphere had evolved, and were global in scope.     

Endospore abundance, microbial growth and necromass turnover in deep sub-seafloor sediment

Two decades of scientific ocean drilling have demonstrated widespread microbial life in deep sub-seafloor sediment, and surprisingly high microbial-cell numbers. Despite the ubiquity of life in the deep biosphere, the large community sizes and the low energy fluxes in this vast buried ecosystem are not yet understood. It is not known whether organisms of the deep biosphere are specifically adapted to extremely low energy fluxes or whether most of the observed cells are in a dormant, spore-like state. Here we apply a new approach--the D:L-amino-acid model--to quantify the distributions and turnover times of living microbial biomass, endospores and microbial necromass, as well as to determine their role in the sub-seafloor carbon budget. The approach combines sensitive analyses of unique bacterial markers (muramic acid and D-amino acids) and the bacterial endospore marker, dipicolinic acid, with racemization dynamics of stereo-isomeric amino acids. Endospores are as abundant as vegetative cells and microbial activity is extremely low, leading to microbial biomass turnover times of hundreds to thousands of years. We infer from model calculations that biomass production is sustained by organic carbon deposited from the surface photosynthetic world millions of years ago and that microbial necromass is recycled over timescales of hundreds of thousands of years.     

Biological activity in the deep subsurface and the origin of heavy oil

At temperatures up to about 80 degrees C, petroleum in subsurface reservoirs is often biologically degraded, over geological timescales, by microorganisms that destroy hydrocarbons and other components to produce altered, denser 'heavy oils'. This temperature threshold for hydrocarbon biodegradation might represent the maximum temperature boundary for life in the deep nutrient-depleted Earth. Most of the world's oil was biodegraded under anaerobic conditions, with methane, a valuable commodity, often being a major by-product, which suggests alternative approaches to recovering the world's vast heavy oil resource that otherwise will remain largely unproduced.     

Biodegradation of oil in uplifted basins prevented by deep-burial sterilization.

Biodegradation of crude oil by bacterial activity--which has occurred in the majority of the Earth's oil reserves--is known to reduce greatly the quality of petroleum in reservoirs. For economically successful prospecting for oil, it is therefore important to understand the processes and conditions in geological formations that lead to oil biodegradation. Although recent studies speculate that bacterial activity can potentially occur up to temperatures as high as 150 degrees C (refs 3, 4), it is generally accepted that effective petroleum biodegradation over geological timescales generally occurs in reservoirs with temperatures below 80 degrees C (ref. 2). This appears, however, to be at odds with the observation that non-degraded oils can still be found in reservoirs below this temperature. Here we compile data regarding the extent of oil biodegradation in several oil reservoirs, and find that the extensive occurrence of non-biodegraded oil in shallow, cool basins is restricted to those that have been uplifted from deeper, hotter regions of the Earth. We suggest that these petroleum reservoirs were sterilized by heating to a temperature around 80-90 degrees C during deep burial, inactivating hydrocarbon-degrading organisms that occur in the deep biosphere. Even when such reservoirs are subsequently uplifted to much cooler regions and filled with oil, degradation does not occur, implying that the sterilized sediments are not recolonized by hydrocarbon-degrading bacteria.     

Major viral impact on the functioning of benthic deep-sea ecosystems

Viruses are the most abundant biological organisms of the world's oceans. Viral infections are a substantial source of mortality in a range of organisms-including autotrophic and heterotrophic plankton-but their impact on the deep ocean and benthic biosphere is completely unknown. Here we report that viral production in deep-sea benthic ecosystems worldwide is extremely high, and that viral infections are responsible for the abatement of 80% of prokaryotic heterotrophic production. Virus-induced prokaryotic mortality increases with increasing water depth, and beneath a depth of 1,000 m nearly all of the prokaryotic heterotrophic production is transformed into organic detritus. The viral shunt, releasing on a global scale approximately 0.37-0.63 gigatonnes of carbon per year, is an essential source of labile organic detritus in the deep-sea ecosystems. This process sustains a high prokaryotic biomass and provides an important contribution to prokaryotic metabolism, allowing the system to cope with the severe organic resource limitation of deep-sea ecosystems. Our results indicate that viruses have an important role in global biogeochemical cycles, in deep-sea metabolism and the overall functioning of the largest ecosystem of our biosphere.     

Terminal Proterozoic reorganization of biogeochemical cycles

The Proterozoic aeon (2,500-540 million years ago) saw episodic increases in atmospheric oxygen content, the evolution of multicellular life and, at its close, an enormous radiation of animal diversity. These profound biological and environmental changes must have been linked, but the underlying mechanisms have been obscure. Here we show that hydrocarbons extracted from Proterozoic sediments in several locations worldwide are derived mainly from bacteria or other heterotrophs rather than from photosynthetic organisms. Biodegradation of algal products in sedimenting matter was therefore unusually complete, indicating that organic material was extensively reworked as it sank slowly through the water column. We propose that a significant proportion of this reworking will have been mediated by sulphate-reducing bacteria, forming sulphide. The production of sulphide and consumption of oxygen near the ocean surface will have inhibited transport of O2 to the deep ocean. We find that preservation of algal-lipid skeletons improves at the beginning of the Cambrian, reflecting the increase in transport by rapidly sinking faecal pellets. We suggest that this rapid removal of organic matter will have increased oxygenation of surface waters, leading to a descent of the O2-sulphide interface to the sea floor and to marked changes in the marine environment, ultimately contributing to the Cambrian radiation.     

Scaling metabolism from organisms to ecosystems

Understanding energy and material fluxes through ecosystems is central to many questions in global change biology and ecology. Ecosystem respiration is a critical component of the carbon cycle and might be important in regulating biosphere response to global climate change. Here we derive a general model of ecosystem respiration based on the kinetics of metabolic reactions and the scaling of resource use by individual organisms. The model predicts that fluxes of CO2 and energy are invariant of ecosystem biomass, but are strongly influenced by temperature, variation in cellular metabolism and rates of supply of limiting resources (water and/or nutrients). Variation in ecosystem respiration within sites, as calculated from a network of CO2 flux towers, provides robust support for the model's predictions. However, data indicate that variation in annual flux between sites is not strongly dependent on average site temperature or latitude. This presents an interesting paradox with regard to the expected temperature dependence. Nevertheless, our model provides a basis for quantitatively understanding energy and material flux between the atmosphere and biosphere.     

Anaerobic hydrocarbon biodegradation in deep subsurface oil reservoirs

Biodegradation of crude oil in subsurface petroleum reservoirs is an important alteration process with major economic consequences. Aerobic degradation of petroleum hydrocarbons at the surface is well documented and it has long been thought that the flow of oxygen- and nutrient-bearing meteoric waters into reservoirs was necessary for in-reservoir petroleum biodegradation. The occurrence of biodegraded oils in reservoirs where aerobic conditions are unlikely, together with the identification of several anaerobic microorganisms in oil fields and the discovery of anaerobic hydrocarbon biodegradation mechanisms, suggests that anaerobic degradation processes could also be responsible. The extent of anaerobic hydrocarbon degradation processes in the world's deep petroleum reservoirs, however, remains strongly contested. Moreover, no organism has yet been isolated that has been shown to degrade hydrocarbons under the conditions found in deep petroleum reservoirs. Here we report the isolation of metabolites indicative of anaerobic hydrocarbon degradation from a large fraction of 77 degraded oil samples from both marine and lacustrine sources from around the world, including the volumetrically important Canadian tar sands. Our results therefore suggest that anaerobic hydrocarbon degradation is a common process in biodegraded subsurface oil reservoirs.     

Early Pleistocene formation of the asymmetric east-west pattern of upper water structure in the equatorial Pacific Ocean

Surface-and subsurface-dwelling planktonic foraminifera from the upper 43 m of Hole A at the Ocean Drilling Program (ODP) Site 807,which was recovered from the western Pacific warm pool during ODP Leg 130,were analyzed for stable oxygen and carbon isotopes.By comparing these results with data from ODP Site 851 in the eastern equatorial Pacific,this study has reconstructed the paleoceanographic changes in upper ocean waters in the equatorial Pacific since 2.5 Ma.During the period from 1.6-1.4 Ma,the oxygen isotopes of surface and subsurface waters were found to markedly change in the western and eastern equatorial Pacific,further confirming the final formation of the well-defined asymmetric east-west (E-W) pattern at that time.This feature was similar to the zonal temperature gradient (sea surface temperature is higher in the west and lower in the east) and the asymmetric upper water structure (thermocline depth is deeper in the west and shallower in the east) in the modern equatorial Pacific.The zonal gradient change of subsurface water δ18O was greater than that of surface water δ18O,indicating that the formation of the asymmetric E-W pattern in the equatorial Pacific should be much more related to the shoaled thermocline and markedly decreased subsurface water temperature in the eastern equatorial Pacific.Moreover,since ～1.6 Ma,the carbon isotopic differences between surface and subsurface waters clearly decreased in the equatorial Pacific,and their long-term eccentricity periods changed from 400 ka to ～500 ka,reflecting the reorganization of the ocean carbon reservoir.This probably resulted from the deep water reorganization in the Southern Ocean at that time and its enhanced influence on the tropical Pacific (especially subsurface water).Our study demonstrates that the tropical ocean plays an important role in global climate change.     

The impact of ocean warming on marine organisms

Since the beginning of the Industrial Revolution during the late eighteenth to the early nineteenth centuries, there has been rapidly increasing release of greenhouse gases, notably CO₂, into the atmosphere. As a consequence of this atmospheric change, the Earth’s average surface temperature has increased by approximately 0.6 °C over the last 100 years. The rate of release of greenhouse gases continues to increase, and global surface temperature rose by approximately 0.2 °C per decade in the last 30 years, a rate that is greater than at any other time during the last 1,000 years. The wide-ranging effects of these increases in greenhouse gases and temperature on the biosphere are subject to intense scientific study. Much has been learned, but much more needs to be elucidated, if we are to predict how terrestrial and aquatic ecosystems will be affected by global change. This brief review focuses on the marine environment and offers a concise summary of some of the important advances in our knowledge about the impacts of global change, including physical and chemical changes of the ocean, as well as the impact of ocean warming on marine organisms. Our analysis also points out areas where critical new information is needed if we are to predict the future of marine ecosystems in a warming world with accuracy.     

First insights into the biodiversity and biogeography of the Southern Ocean deep sea

Shallow marine benthic communities around Antarctica show high levels of endemism, gigantism, slow growth, longevity and late maturity, as well as adaptive radiations that have generated considerable biodiversity in some taxa. The deeper parts of the Southern Ocean exhibit some unique environmental features, including a very deep continental shelf and a weakly stratified water column, and are the source for much of the deep water in the world ocean. These features suggest that deep-sea faunas around the Antarctic may be related both to adjacent shelf communities and to those in other oceans. Unlike shallow-water Antarctic benthic communities, however, little is known about life in this vast deep-sea region. Here, we report new data from recent sampling expeditions in the deep Weddell Sea and adjacent areas (748-6,348 m water depth) that reveal high levels of new biodiversity; for example, 674 isopods species, of which 585 were new to science. Bathymetric and biogeographic trends varied between taxa. In groups such as the isopods and polychaetes, slope assemblages included species that have invaded from the shelf. In other taxa, the shelf and slope assemblages were more distinct. Abyssal faunas tended to have stronger links to other oceans, particularly the Atlantic, but mainly in taxa with good dispersal capabilities, such as the Foraminifera. The isopods, ostracods and nematodes, which are poor dispersers, include many species currently known only from the Southern Ocean. Our findings challenge suggestions that deep-sea diversity is depressed in the Southern Ocean and provide a basis for exploring the evolutionary significance of the varied biogeographic patterns observed in this remote environment.     

东南印度洋海域冬季水文特征及其水团分析

为了研究东南印度洋海域冬季水文特征及其水团结构,利用澳大利亚海洋与南极研究所2012-2013年南半球冬季在东南印度洋观测得到的温度、盐度和溶解氧的资料进行分析。结果表明:表层温度呈现明显的北高南低态势,次表层均存在较强的温度、盐度、密度跃层,且均呈现出高/低盐带与高/低溶解氧带,高盐带在经向断面上最为明显;在105°E断面上存在显著的中尺度现象,且呈现上下层暖、冷涡叠置的分布特征,根据温度、盐度及溶解氧的特性,除南印度洋中央水和南极中层水外,分别将研究海域其他水团命名为副热带东南印度洋表层水、副热带东南印度洋次表层水、副热带东南印度洋模态水、副热带东南印度洋中层水、副热带东南印度洋深层水和副热带东南印度洋底层水,并给出各自的特性指标。     

Evolution of plant reproduction: From fusion and dispersal to interaction and communication

Based on the existing data concerning the evolution of the sexual reproduction, it is argued that the processes of sex differentiation and interactions play a key role in evolution. From the beginning environment and organism are unified. In a changing dynamic environment life originates and the interaction between life and environment develops from simple to more complex organisms. Sexual reproduction is introduced after the origin of meiosis and is a key process in evolution. The asexual reproduction process prepares to dispersal. Sexual reproduction process adds the genome renewal and the gamete-gamete interaction. Reproduction and dispersal are connected and the process of reproduction has similarities between asexual and sexual reproduction. Unicellular algae develop the physiological and morphological sex differentiation. Sex differentiation is connected with the way of dispersal. The step to multicellular plants introduces cell isolation after meiosis and by the stay on the mother plant within a cell or organ, plant-cell apoplastic interaction originates and by prolonged stay the plant-plant interaction. This stay influences the type of dispersal. A life cycle with alternation of generations and two moments of dispersal permits plants to go on land. In ferns a shift in the moment of sex differentiation to meiospore happens and the stay of the macrospore leads to the seed plants. In water all types of sexual reproduction, interactions and the alternation of generations are prepared and these are used to conquest land. On land the biotic dispersal is realized. The phylogeny of sexual reproduction reveals that the sex differentiation and interaction are the main causes in the evolution of sexual reproduction. Sexual reproduction shows interactions during gamete fusion, between organism and environment and in multicellular plants between organisms. With respect to other types of interaction as in symbiosis or the nutrient chain, interaction is considered as an important action which is based on a persisting cooperation and points to a push during evolution. The push is expressed as communication： the driving force in the evolution. Based on the interactions between organisms and interactions between organisms and the dynamic environment, communication is considered as a driving force leading to the evolution as explained in the development of plant reproduction. Consequences for reproduction, its regulation and the process of evolution are discussed.     

Oxidation of the Ediacaran ocean

Oxygenation of the Earth's surface is increasingly thought to have occurred in two steps. The first step, which occurred approximately 2,300 million years (Myr) ago, involved a significant increase in atmospheric oxygen concentrations and oxygenation of the surface ocean. A further increase in atmospheric oxygen appears to have taken place during the late Neoproterozoic period ( approximately 800-542 Myr ago). This increase may have stimulated the evolution of macroscopic multicellular animals and the subsequent radiation of calcified invertebrates, and may have led to oxygenation of the deep ocean. However, the nature and timing of Neoproterozoic oxidation remain uncertain. Here we present high-resolution carbon isotope and sulphur isotope records from the Huqf Supergroup, Sultanate of Oman, that cover most of the Ediacaran period (approximately 635 to approximately 548 Myr ago). These records indicate that the ocean became increasingly oxygenated after the end of the Marinoan glaciation, and they allow us to identify three distinct stages of oxidation. When considered in the context of other records from this period, our data indicate that certain groups of eukaryotic organisms appeared and diversified during the second and third stages of oxygenation. The second stage corresponds with the Shuram excursion in the carbon isotope record and seems to have involved the oxidation of a large reservoir of organic carbon suspended in the deep ocean, indicating that this event may have had a key role in the evolution of eukaryotic organisms. Our data thus provide new insights into the oxygenation of the Ediacaran ocean and the stepwise restructuring of the carbon and sulphur cycles that occurred during this significant period of Earth's history.     

Chance and necessity: the evolution of morphological complexity and diversity

The primary foundation for contemplating the possible forms of life elsewhere in the Universe is the evolutionary trends that have marked life on Earth. For its first three billion years, life on Earth was a world of microscopic forms, rarely achieving a size greater than a millimetre or a complexity beyond two or three cell types. But in the past 600 million years, the evolution of much larger and more complex organisms has transformed the biosphere. Despite their disparate forms and physiologies, the evolution and diversification of plants, animals, fungi and other macroforms has followed similar global trends. One of the most important features underlying evolutionary increases in animal and plant size, complexity and diversity has been their modular construction from reiterated parts. Although simple filamentous and spherical forms may evolve wherever cellular life exists, the evolution of motile, modular mega-organisms might not be a universal pattern.     

Deep sub-seafloor prokaryotes stimulated at interfaces over geological time

The sub-seafloor biosphere is the largest prokaryotic habitat on Earth but also a habitat with the lowest metabolic rates. Modelled activity rates are very low, indicating that most prokaryotes may be inactive or have extraordinarily slow metabolism. Here we present results from two Pacific Ocean sites, margin and open ocean, both of which have deep, subsurface stimulation of prokaryotic processes associated with geochemical and/or sedimentary interfaces. At 90 m depth in the margin site, stimulation was such that prokaryote numbers were higher (about 13-fold) and activity rates higher than or similar to near-surface values. Analysis of high-molecular-mass DNA confirmed the presence of viable prokaryotes and showed changes in biodiversity with depth that were coupled to geochemistry, including a marked community change at the 90-m interface. At the open ocean site, increases in numbers of prokaryotes at depth were more restricted but also corresponded to increased activity; however, this time they were associated with repeating layers of diatom-rich sediments (about 9 Myr old). These results show that deep sedimentary prokaryotes can have high activity, have changing diversity associated with interfaces and are active over geological timescales.     

Adherens junctions and beta-catenin-mediated cell signalling in a non-metazoan organism

Mechanical forces between cells have a principal role in the organization of animal tissues. Adherens junctions are an important component of these tissues, connecting cells through their actin cytoskeleton and allowing the assembly of tensile structures. At least one adherens junction protein, beta-catenin, also acts as a signalling molecule, directly regulating gene expression. To date, adherens junctions have only been detected in metazoa, and therefore we looked for them outside the animal kingdom to examine their evolutionary origins. The non-metazoan Dictyostelium discoideum forms a multicellular, differentiated structure. Here we describe the discovery of actin-associated intercellular junctions in Dictyostelium. We have isolated a gene encoding a beta-catenin homologue, aardvark, which is a component of the junctional complex, and, independently, is required for cell signalling. Our discovery of adherens junctions outside the animal kingdom shows that the dual role of beta-catenin in cell-cell adhesion and cell signalling evolved before the origins of metazoa.     

Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-gamma and PTEN

Wound healing is essential for maintaining the integrity of multicellular organisms. In every species studied, disruption of an epithelial layer instantaneously generates endogenous electric fields, which have been proposed to be important in wound healing. The identity of signalling pathways that guide both cell migration to electric cues and electric-field-induced wound healing have not been elucidated at a genetic level. Here we show that electric fields, of a strength equal to those detected endogenously, direct cell migration during wound healing as a prime directional cue. Manipulation of endogenous wound electric fields affects wound healing in vivo. Electric stimulation triggers activation of Src and inositol-phospholipid signalling, which polarizes in the direction of cell migration. Notably, genetic disruption of phosphatidylinositol-3-OH kinase-gamma (PI(3)Kgamma) decreases electric-field-induced signalling and abolishes directed movements of healing epithelium in response to electric signals. Deletion of the tumour suppressor phosphatase and tensin homolog (PTEN) enhances signalling and electrotactic responses. These data identify genes essential for electrical-signal-induced wound healing and show that PI(3)Kgamma and PTEN control electrotaxis.     

GCr15钢超高周的疲劳行为

以GCr15钢为试验材料进行旋转弯曲超高周疲劳行为的试验研究,用电子显微镜对试样断口进行观察.结果表明:疲劳裂纹的萌生机制可以分为两种,一种为表面裂纹萌生机制,发生在高应力幅短寿命区,是由试样表面晶体滑移或表面夹杂引起的;另一种为内部裂纹萌生机制,发生在低应力幅长寿命区,是由试样内部的非金属夹杂物引起的.通过对试验结果的分析和处理,描绘出了GCr15钢的S-N曲线.通过对裂纹萌生位置处尺寸参数的计算和评估,阐述了裂纹萌生于内部的破坏机理,提出了基于裂纹尺寸参数的超高周疲劳极限的推定方法.