IODP Expedition 329: Life and Habitability Beneath the Seafloor of the South Pacific Gyre

Integrated Ocean Drilling Program (IODP) Expedition 329 made major strides toward fulfilling its objectives. Shipboard studies documented (1) fundamental aspects of habitability and life in this very low activity subseafloor sedimentary ecosystem and (2) first-order patterns of habitability within the igneous basement. A broad range of postexpedition studies will complete the expedition objectives. Throughout the South Pacific Gyre (SPG; Sites U1365–U1370), dissolved oxygen and nitrate are present throughout the entire sediment sequence, and sedimentary microbial cell counts are lower than at all previously drilled IODP/ Ocean Drilling Program (ODP)/Deep Sea Drilling Program (DSDP) sites. In contrast, at Site U1371 in the upwelling zone just south of the gyre, detectable oxygen and nitrate are limited to the top and bottom of the sediment column, manganese reduction is a prominent electron-accepting process, and cell concentrations are higher than at the same depths in the SPG sites throughout the sediment column. Geographic variation in subseafloor profiles of dissolved and solid-phase chemicals are consistent with the magnitude of organic-fueled subseafloor respiration declining from outside the gyre to the gyre center. Chemical profiles in the sedimentary pore water and secondary mineral distributions in the basaltic basement indicate that basement alteration continues on the timescale of formation fluid replacement, even at the sites with the oldest basement (84–120 Ma at Sites U1365 and U1366)

Roles of Bone Morphogenetic Protein-6 in Aldosterone Regulation by Adrenocortical Cells

Aldosterone production occurs in the adrenal cortex, and is regulated primarily by angiotensin II (Ang II), potassium and adrenocorticotropin (ACTH). In the presence of the aldosterone stimulators, steroidogenesis is further governed by local autocrine and/or paracrine factors in the adrenal cortex. We reported the presence of functional bone morphogenetic protein (BMP) system in the adrenal cortex and also demonstrated that BMP-6 increases Ang II-induced aldosterone production, which could be involved in the "aldosterone breakthrough" phenomenon. Aldosterone breakthrough is the phenomenon by which circulating aldosterone concentrations increase above pre-treatment levels after long-term therapy with ACE inhibitors or Ang II type 1 receptor antagonists (ARB). This phenomenon may lead to important clinical consequences since increased aldosterone in a high-salt state facilitates cardiovascular and renal damage in hypertensive patients. We found that long-term ARB treatment reverses the reduction of aldosterone synthesis by adrenocortical cells, thereby causing "cellular aldosterone breakthrough". The availability of BMP-6 in the adrenal cortex may be at least partly involved in the occurrence of cellular escape from aldosterone suppression under chronic treatment with ARB

Bioelectrochemical Stimulation of Electromethanogenesis at a Seawater-Based Subsurface Aquifer in a Natural Gas Field

In subsurface anoxic environments, microbial communities generally produce methane as an end-product to consume organic compounds. This metabolic function is a source of biogenic methane in coastal natural gas aquifers, submarine mud volcanoes, and methane hydrates. Within the methanogenic communities, hydrogenotrophic methanogens, and syntrophic bacteria are converting volatile fatty acids to methane syntrophically via interspecies hydrogen transfer. Recently, direct interspecies electron transfer (DIET) between fermentative/syntrophic bacteria and electrotrophic methanogens has been proposed as an effective interspecies metabolite transfer process to enhance methane production. In this study, in order to stimulate the DIET-associated methanogenic process at deep biosphere-aquifer systems in a natural gas field, we operated a bioelectrochemical system (BES) to apply voltage between an anode and a cathode. Two single-chamber BESs were filled with seawater-based formation water collected from an onshore natural gas well, repeatedly amended with acetate, and operated with 600 mV between electrodes for 21 months, resulting in a successful conversion of acetate to methane via electrical current consumption. One reactor yielded a stable current of ~200 mA/m2 with a coulombic efficiency (CE) of >90%; however, the other reactor, which had been incidentally disconnected for 3 days, showed less electromethanogenic activity with a CE of only ~10%. The 16S rRNA gene-based community analyses showed that two methanogenic archaeal families, Methanocalculaceae and Methanobacteriaceae, were abundant in cathode biofilms that were mainly covered by single-cell-layered biofilm, implicating them as key players in the electromethanogenesis. In contrast, family Methanosaetaceae was abundant at both electrodes and the electrolyte suspension only in the reactor with less electromethanogenesis, suggesting this family was not involved in electromethanogenesis and became abundant only after the no-electron-flow event. The anodes were covered by thick biofilms with filamentous networks, with the family Desulfuromonadaceae dominating in the early stage of the operation. The family Geobacteraceae (mainly genus Geoalkalibacter) became dominant during the longer-term operation, suggesting that these families were correlated with electrode-respiring reactions. These results indicate that the BES reactors with voltage application effectively activated a subsurface DIET-related methanogenic microbiome in the natural gas field, and specific electrogenic bacteria and electromethanogenic archaea were identified within the anode and/or cathode biofilms

Activities of bone morphogenetic proteins in prolactin regulation by somatostatin analogs in rat pituitary GH3 cells

Involvement of the pituitary BMP system in the modulation of prolactin (PRL) secretion regulated by somatostatin analogs, including octreotide (OCT) and pasireotide (SOM230), and a dopamine agonist, bromocriptine (BRC), was examined in GH3 cells. GH3 cells are rat pituitary somato-lactotrope tumor cells that express somatostatin receptors (SSTRs) and BMP system molecules including BMP-4 and -6. Treatment with BMP-4 and -6 increased PRL and cAMP secretion by GH3 cells. The BMP-4 effects were neutralized by adding a BMP-binding protein Noggin. These findings suggest the activity of endogenous BMPs in augmenting PRL secretion by GH3 cells. BRC and SOM230 reduced PRL secretion, but OCT failed to reduce the PRL level. In GH3 cells activated by forskolin, BRC suppressed forskolin-induced PRL secretion with reduction in cAMP levels. OCT did not affect forskolin-induced PRL level, while SOM230 reduced PRL secretion and PRL mRNA expression induced by forskolin. BMP-4 treatment enhanced the reducing effect of SOM230 on forskolin-induced PRL level while BMP-4 did not affect the effects of OCT or BRC. Noggin treatment had no significant effect on the BRC actions reducing PRL levels by GH3 cells. However, in the presence of Noggin, OCT elicited an inhibitory effect on forskolin-induced PRL secretion and PRL mRNA expression, whereas the SOM230 effect on PRL reduction was in turn impaired. It was further found that BMP-4 and -6 suppressed SSTR-2 but increased SSTR-5 mRNA expression of GH3 cells. These findings indicate that Noggin rescues SSTR-2 but downregulates SSTR-5 by neutralizing endogenous BMP actions, leading to an increase in OCT sensitivity and a decrease in SOM230 sensitivity of GH3 cells. In addition, BMP signaling was facilitated in GH3 cells treated with forskolin. Collectively, these findings suggest that BMPs elicit differential actions in the regulation of PRL release dependent on cellular cAMP-PKA activity. BMPs may play a key role in the modulation of SSTR sensitivity of somato-lactotrope cells in an autocrine/paracrine manner

IODP Advances in the Understanding of Subseafloor Life

The most recent decadal phase of scientific ocean drilling through the International Ocean Discovery Program (IODP) has resulted in paradigm-shifting understanding of life below the seafloor. Enabled by new drilling and coring approaches, cutting-edge methodologies, and novel observatory science, IODP expeditions have significantly advanced understanding of the amount and diversity of subseafloor life, the metabolic strategies that this life uses to survive under extreme energy limitation, and consequences of this life for the Earth system. Here, we summarize highlights from recent IODP expeditions focused on life beneath the seafloor and emphasize remaining major science challenges in investigating the form and function of life in this environment

Atribacteria from the Subseafloor Sedimentary Biosphere Disperse to the Hydrosphere through Submarine Mud Volcanoes

Submarine mud volcanoes (SMVs) are formed by muddy sediments and breccias extruded to the seafloor from a source in the deep subseafloor and are characterized by the discharge of methane and other hydrocarbon gasses and deep-sourced fluids into the overlying seawater. Although SMVs act as a natural pipeline connecting the Earth’s surface and subsurface biospheres, the dispersal of deep-biosphere microorganisms and their ecological roles remain largely unknown. In this study, we investigated the microbial communities in sediment and overlying seawater at two SMVs located on the Ryukyu Trench off Tanegashima Island, southern Japan. The microbial communities in mud volcano sediments were generally distinct from those in the overlying seawaters and in the well-stratified Pacific margin sediments collected at the Peru Margin, the Juan de Fuca Ridge flank off Oregon, and offshore of Shimokita Peninsula, northeastern Japan. Nevertheless, in-depth analysis of different taxonomic groups at the sub-species level revealed that the taxon affiliated with Atribacteria, heterotrophic anaerobic bacteria that typically occur in organic-rich anoxic subseafloor sediments, were commonly found not only in SMV sediments but also in the overlying seawater. We designed a new oligonucleotide probe for detecting Atribacteria using the catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). CARD-FISH, digital PCR and sequencing analysis of 16S rRNA genes consistently showed that Atribacteria are abundant in the methane plumes of the two SMVs (0.58 and 1.5 × 104 cells/mL, respectively) but not in surrounding waters, suggesting that microbial cells in subseafloor sediments are dispersed as “deep-biosphere seeds” into the ocean. These findings may have important implications for the microbial transmigration between the deep subseafloor biosphere and the hydrosphere

Optimization of PID parameters for hydraulic positioning system utilizing variable weight Grey-Taguchi and particle swarm optimization

Controller that uses PID parameters requires a good tuning method in order to improve the control system performance. Especially on hydraulic positioning system that is highly nonlinear and difficult to be controlled whereby PID parameters needs to be tuned to obtain optimum performance criteria. Tuning PID control method is divided into two namely the classical methods and the methods of artificial intelligence. Particle swarm optimization algorithm (PSO) is one of the artificial intelligence methods. Previously, researchers had integrated PSO algorithms in the PID parameter tuning process. This research aims to improve the PSO-PID tuning algorithms by integrating the tuning process with the Variable Weight Grey-Taguchi Design of Experiment (DOE) method. This is done by conducting the DOE on the two PSO optimizing parameters: the limit of change in particle velocity and the weight distribution factor. Computer simulations and physical experiments were conducted by using the proposed PSO-PID with the Variable Weight Grey-Taguchi DOE and the classical Ziegler-Nichols methods. They are implemented on the hydraulic positioning system. Simulation results show that the proposed PSO-PID with the Variable Weight Grey-Taguchi DOE has reduced the rise time by 48.13% and settling time by 48.57% compared to the Ziegler-Nichols method. Physical experiment results also show that the proposed PSO-PID with the Variable Weight Grey-Taguchi DOE tuning responds better than Ziegler-Nichols tuning. In conclusion, this research has improved the PSO-PID parameter by applying the PSO-PID algorithm together with the Variable Weight Grey-Taguchi DOE method as a good tuning method in the hydraulic positioning system

Microbial Metabolism and Community Dynamics in Hydraulic Fracturing Fluids Recovered From Deep Hydrocarbon-Rich Shale

Hydraulic fracturing is a prominent method of natural gas production that uses injected, high-pressure fluids to fracture low permeability, hydrocarbon rich strata such as shale. Upon completion of a well, the fluid returns to the surface (produced water) and contains natural gas, subsurface constituents, and microorganisms (Barbot et al., 2013; Daly et al., 2016). While the microbial community of the produced fluids has been studied in multiple gas wells, the activity of these microorganisms and their relation to biogeochemical activity is not well understood. In this experiment, we supplemented produced fluid with 13C-labeled carbon sources (glucose, acetate, bicarbonate, methanol, or methane), and 15N-labeled ammonium chloride in order to isotopically trace microbial activity over multiple day in anoxic incubations. Nanoscale secondary ion mass spectrometry (NanoSIMS) was used to generate isotopic images of 13C and 15N incorporation in individual cells, while isotope ratio monitoring–gas chromatography–mass spectrometry (IRM–GC–MS) was used to measure 13CO2, and 13CH4 as metabolic byproducts. Glucose, acetate, and methanol were all assimilated by microorganisms under anoxic conditions. 13CO2 production was only observed with glucose as a substrate indicating that catabolic activity was limited to this condition. The microbial communities observed at 0, 19, and 32 days of incubation did not vary between different carbon sources, were low in diversity, and composed primarily of the class Clostridia. The primary genera detected in the incubations, Halanaerobium and Fusibacter, are known to be adapted to harsh physical and chemical conditions consistent with those that occur in the hydrofracturing environment. This study provides evidence that microorganisms in produced fluid are revivable in laboratory incubations and retained the ability to metabolize added carbon and nitrogen substrates