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Agarwal, V, Li J, Rahman I, Borgen M, Aluwihare LI, Biggs JS, Paul VJ, Moore BS.  2015.  Complexity of naturally produced polybrominated diphenyl ethers revealed via mass spectrometry. Environmental Science & Technology. 49:1339-1346.   10.1021/es505440j   AbstractWebsite

Polybrominated diphenyl ethers (PBDEs) are persistent and bioaccumulative anthropogenic and natural chemicals that are broadly distributed in the marine environment. PBDEs are potentially toxic due to inhibition of various mammalian signaling pathways and enzymatic reactions. PBDE isoforms vary in toxicity in accordance with structural differences, primarily in the number and pattern of hydroxyl moieties afforded upon a conserved core structure. Over four decades of isolation and discovery-based efforts have established an impressive repertoire of natural PBDEs. Based on our recent reports describing the bacterial biosyntheses of PBDEs, we predicted the presence of additional classes of PBDEs to those previously identified from marine sources. Using mass spectrometry and NMR spectroscopy, we now establish the existence of new structural classes of PBDEs in marine sponges. Our findings expand the chemical space explored by naturally produced PBDEs, which may inform future environmental toxicology studies. Furthermore, we provide evidence for iodinated PBDEs and direct attention toward the contribution of promiscuous halogenating enzymes in further expanding the diversity of these polyhalogenated marine natural products.

Aluwihare, LI, Repeta DJ, Chen RF.  2002.  Chemical composition and cycling of dissolved organic matter in the Mid-Atlantic Bight. Deep-Sea Research Part Ii-Topical Studies in Oceanography. 49:4421-4437.   10.1016/s0967-0645(02)00124-8   AbstractWebsite

This study focuses on the chemical characterization of high molecular-weight dissolved organic matter (HMW DOM) isolated from the Middle Atlantic Bight in April 1994 and March 1996. Using proton nuclear magnetic resonance spectroscopy ((HNMR)-H-1) and monosaccharide analysis we compared both spatial and temporal variations in the chemical structure of HMW DOM across this region. Our analyses support the presence of at least two compositionally distinct components to HMW DOM. The major component is acyl polysaccharide (APS), a biopolymer rich in carbohydrates, acetate and lipid, accounting for between 50% and 80% of the total high molecular-weight dissolved organic carbon (HMW DOC) in surface samples. APS is most abundant in fully marine, surface-water samples, and is a product of autochthonous production. Organic matter with spectral properties characteristic of humic substances is the second major component of HMW DOM. Humic substances are most abundant (up to 49% of the total carbon) in samples collected from estuaries, near the coast, and in deep water, suggesting both marine and perhaps terrestrial sources. Radiocarbon analyses of neutral monosaccharides released by the hydrolysis of APS have similar and modern (average 71parts per thousand) Delta(14)C values. Radiocarbon data support our suggestion that these sugars occur as part of a common macromolecule, with an origin via recent biosynthesis. Preliminary radiocarbon data for total neutral monosaccharides isolated from APS at 300 and 750m show this fraction to be substantially enriched relative to total HMW DOC and DOC. The relatively enriched radiocarbon values of APS at depth suggest APS is rapidly transported into the deep ocean. (C) 2002 Elsevier Science Ltd. All rights reserved.

Aluwihare, LI, Repeta DJ, Chen RF.  1997.  A major biopolymeric component to dissolved organic carbon in surface sea water. Nature. 387:166-169.   10.1038/387166a0   AbstractWebsite

Organic carbon dissolved in sea water is an important component of the global carbon cycle(1). Concentrations of dissolved organic carbon (DOC) in the ocean's surface mixed layer are at least twice those in the deep sea(2,3), because of the production of soluble carbon compounds by marine algae in the euphotic zone(4,5). But very little is known about the chemical composition of DOC, and the connection between photosynthetic production and DOC accumulation is not well understood(6,7). Here we report the chemical characterization of macromolecular DOC at several sites in the Atlantic and Pacific oceans. Neutral sugars, acetate and lipids show similar distributions, suggesting that these constituents are linked together in a common macromolecular structure. Chemical linkage patterns between the oligosaccharide portions of dissolved organic matter subjected to ultrafiltration are highly specific, with little variation between ocean basins. We show that laboratory culture experiments on the decomposition of algal exudate produce macromolecular organic matter with similar compositions and linkage characteristics. We propose that a significant fraction of DOC in sea surface water consists of structurally related and biosynthetically derived acyl oligosaccharides that persist after more labile organic matter has been degraded.

Aluwihare, LI, Repeta DJ, Pantoja S, Johnson CG.  2005.  Two chemically distinct pools of organic nitrogen accumulate in the ocean. Science. 308:1007-1010.   10.1126/science.1108925   AbstractWebsite

The chemical dynamics of marine dissolved organic nitrogen (DON), a reservoir featuring surface accumulations even in areas where nitrogen limits productivity, have yet to be resolved. We exploited differences in the acid lability of amide bonds within high-molecular-weight (HMW) DON to show that vertical DON profiles result in part from the presence of two chemically distinct pools of amide. Half of HMWDON in surface waters is present as N-acetyl amino polysaccharides. In contrast, nearly all deep-sea HMWDON, and therefore, most HMWDON, is present in amides that resist both chemical hydrolysis and biological degradation.

Aluwihare, LI, Repeta DJ.  1999.  A comparison of the chemical characteristics of oceanic DOM and extracellular DOM produced by marine algae. Marine Ecology-Progress Series. 186:105-117.   10.3354/meps186105   AbstractWebsite

The chemical characteristics of extracellular high molecular weight (HMW) dissolved organic matter (DOM) from 3 species of marine phytoplankton were compared to HMW DOM in seawater. Thalassiosira weissflogii, Emiliania huxleyi and Phaeocystis sp., were grown in nutrient enriched seawater that had been previously ultrafiltered to remove HMW DOM. The extracellular HMW DOM produced in these cultures was isolated by ultrafiltration and characterized using nuclear magnetic resonance (NMR) spectroscopy, and molecular level analyses. All species exude DOM rich in polysaccharides, and the exudates of T. weissflogii and E. huxleyi closely resemble acyl heteropolysaccharides (APS) previously identified as major constituents of naturally occurring marine HMW DOM. Degradation of the T. weissflogii exudate alters the chemical composition of the DOM, which we attribute to differences in the reactivity of specific polysaccharides. The component within the exudate that most resembles seawater DOM has a slower degradation rate relative to the total polysaccharide fraction. Our study indicates that APS isolated from the surface ocean can have a direct algal source and that APS may accumulate in seawater as a result of its metabolic resistance.

Arakawa, N, Aluwihare L.  2015.  Direct identification of diverse alicyclic terpenoids in Suwannee River fulvic acid. Environmental Science & Technology. 49:4097-4105.   10.1021/es5055176   AbstractWebsite

The chemical complexity of dissolved organic matter (DOM) obstructs our ability to definitively recover source compounds from within DOM, an objective which has the capacity to alter our understanding, of carbon sequestration on a. global scale. To advance compositional studies of DOM we have applied a previously published reduction method to an environmental standard, Suwannee River Fulvic Acid (SRFA). The reduction products, comprising 12% of the prereduced carbon, were then separated by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GCXGC-TOF-MS). estilts indicate that the majority of obsetved reduced compounds corresponded to alicyclic hydrocarbons in the size range C-10 to C-17. Cyclic terpenoids are the only biomolecule class with contiguous, alicyclic carbon backbones of this size. These terpenoid reduction products contain series offset by CH2 and exhibit great isomeric diversity, features previously inferred from ultrahigh resolution mass spectrometry and NMR studies of unreduced SRFA. Reduction of Taxodium leaf litter as a Source material to SRFA confirmed the prevalence of tetpenoids in SRFA and provided insight into the parent compounds that must be diagenetically modified on relatively short time scales. These data corroborate several recent studies that suggest alicydic hydrocarbons to be important components of longer-lived DOM.

Arakawa, N, Aluwihare LI, Simpson AJ, Soong R, Stephens BM, Lane-Coplen D.  2017.  Carotenoids are the likely precursor of a significant fraction of marine dissolved organic matter. Science Advances. 3   10.1126/sciadv.1602976   Abstract

The ocean’s biota sequester atmospheric carbon dioxide (CO2) in part by producing dissolved organic matter (DOM) that persists in the ocean for millennia. This long-term accumulation of carbon may be facilitated by abiotic and biotic production of chemical structures that resist degradation, consequently contributing disproportionately to refractory DOM. Compounds that are selectively preserved in seawater were identified in solid-phase extracted DOM (PPL-DOM) using comprehensive gas chromatography (GC) coupled to mass spectrometry (MS). These molecules contained cyclic head groups that were linked to isoprenoid tails, and their overall structures closely resembled carotenoid degradation products (CDP). The origin of these compounds in PPL-DOM was further confirmed with an in vitro β-carotene photooxidation experiment that generated water-soluble CDP with similar structural characteristics. The molecular-level identification linked at least 10% of PPL-DOM carbon, and thus 4% of total DOM carbon, to CDP. Nuclear magnetic resonance spectra of experimental CDP and environmental PPL-DOM overlapped considerably, which indicated that even a greater proportion of PPL-DOM was likely composed of CDP. The CDP-rich DOM fraction was depleted in radiocarbon (14C age > 1500 years), a finding that supports the possible long-term accumulation of CDP in seawater. By linking a specific class of widespread biochemicals to refractory DOM, this work provides a foundation for future studies that aim to examine how persistent DOM forms in the ocean.

Ball, GI, Xu L, McNichol AP, Aluwihare LI.  2012.  A two-dimensional, heart-cutting preparative gas chromatograph facilitates highly resolved single-compound isolations with utility towards compound-specific natural abundance radiocarbon (C-14) analyses. Journal of Chromatography A. 1220:122-131.   10.1016/j.chroma.2011.11.021   AbstractWebsite

Motivated by the need to develop clean, high purity preparative enrichments of individual compounds for micro-scale compound-specific natural abundance isotope and radiocarbon (C-14) analyses, we describe a new, two-dimensional, heart-cutting, low-bleed, three-oven, single GC preparative system, demonstrate its resolving capabilities as applied to a typically complex environmental sample matrix, and investigate the robustness with which it preserves the authigenic C-13/C-12 and C-14/C-12 ratios of individual compounds it targets for preparative enrichment. The system is comprised of a programmable temperature vaporizing (PTV) inlet, a single GC oven, two modular, door-mounted, resistively heated low thermal mass (LTM) columns, a preparative fraction collector (PFC), and a Deans pneumatic switching device which facilitates heart-cutting between the system's 1 degrees and 2 degrees chromatographic dimensions. Further, the system's inlet and trapping parameters are optimized for the efficient preparative enrichment of the methyl ether and ester derivatives of the lignin phenol compound class. The lignin phenols include such compounds as the vanillyl and syringyl aldehydes, ethanones, and acids and are unrivaled biomarkers of terrestrial organic matter, some of which are also important components of fragrances and flavors. Using this suite of compounds, the suitability of this augmented preparative capillary GC (PCGC) system was investigated for micro-scale compound-specific (CS) stable isotope and natural abundance radiocarbon analyses (RA). Analysis of a >300 injection enrichment scheme reveals the instrument to fractionate C-13 in predictable ways and to preserve the authigenic Delta C-14 of compounds it targets for preparative enrichment to within 6.7 +/- 5.0 parts per thousand, demonstrating the promising new utility of such systems towards micro-scale CSRA investigations for which clean and high resolution separation techniques are prerequisite. (C) 2011 Elsevier B.V. All rights reserved.

Beaupre, SR, Aluwihare L.  2010.  Constraining the 2-component model of marine dissolved organic radiocarbon. Deep-Sea Research Part Ii-Topical Studies in Oceanography. 57:1494-1503.   10.1016/j.dsr2.2010.02.017   AbstractWebsite

Keeling plots of dissolved organic carbon (DOC) concentration and Δ14C depth profiles imply rapid, diapycnal transport of DOC to the meso- and bathypelagic zones, but do not constrain the mechanism of redistribution. We review the 2-component Keeling plot model, and present an alternative formulation explicitly based on homogenization of water parcels. Applying this new model to DOC and dissolved inorganic carbon (DIC) depth profiles suggests that the dominant controls on DOC redistribution differ throughout the water column. DOC concentration and Δ14C gradients were consistent with biogeochemical processing in the epipelagic and advection in the mesopelagic. Vertical gradients in DOC concentration and Δ14C were insufficient for further interpretation in the bathypelagic. Ultimately, additional concurrent measurements of DOC and DIC concentrations and Δ14C values throughout the water column at more locations are needed to constrain the applicability of two-component mixing models to marine DOC.

Close, HG, Shah SR, Ingalls AE, Diefendorf AF, Brodie EL, Hansman RL, Freeman KH, Aluwihare LI, Pearson A.  2013.  Export of submicron particulate organic matter to mesopelagic depth in an oligotrophic gyre. Proceedings of the National Academy of Sciences of the United States of America. 110:12565-12570.   10.1073/pnas.1217514110   AbstractWebsite

Sixty percent of the world ocean by area is contained in oligotrophic gyres [Longhurst A (1995) Prog Oceanog 36:77-16], the biomass of which is dominated by picophytoplankton, including cyanobacteria and picoeukaryotic algae, as well as picoheterotrophs. Despite their recognized importance in carbon cycling in the surface ocean, the role of small cells and their detrital remains in the transfer of particulate organic matter (POM) to the deep ocean remains disputed. Because oligotrophic marine conditions are projected to expand under current climate trends, a better understanding of the role of small particles in the global carbon cycle is a timely goal. Here we use the lipid profiles, radiocarbon, and stable carbon isotopic signatures of lipids from the North Pacific Subtropical Gyre to show that in the surface ocean, lipids from submicron POM (here called extra-small POM) are distinct from larger classes of suspended POM. Remarkably, this distinct extra-small POM signature dominates the total lipids collected at mesopelagic depth, suggesting that the lipid component of mesopelagic POM primarily contains the exported remains of small particles. Transfer of submicron material to mesopelagic depths in this location is consistent with model results that claim the biological origin of exported carbon should be proportional to the distribution of cell types in the surface community, irrespective of cell size [Richardson TL, Jackson GA (2007) Science 315:838-840]. Our data suggest that the submicron component of exported POM is an important contributor to the global biological pump, especially in oligotrophic waters.

Collins, DB, Ault AP, Moffet RC, Ruppel MJ, Cuadra-Rodriguez LA, Guasco TL, Corrigan CE, Pedler BE, Azam F, Aluwihare LI, Bertram TH, Roberts GC, Grassian VH, Prather KA.  2013.  Impact of marine biogeochemistry on the chemical mixing state and cloud forming ability of nascent sea spray aerosol. Journal of Geophysical Research-Atmospheres. 118:8553-8565.   10.1002/jgrd.50598   AbstractWebsite

The composition and properties of sea spray aerosol, a major component of the atmosphere, are often controlled by marine biological activity; however, the scope of impacts that ocean chemistry has on the ability for sea spray aerosol to act as cloud condensation nuclei (CCN) is not well understood. In this study, we utilize a mesocosm experiment to investigate the impact of marine biogeochemical processes on the composition and mixing state of sea spray aerosol particles with diameters<0.2 mu m produced by controlled breaking waves in a unique ocean-atmosphere facility. An increase in relative abundance of a distinct, insoluble organic particle type was observed after concentrations of heterotrophic bacteria increased in the seawater, leading to an 86 +/- 5% reduction in the hygroscopicity parameter () at 0.2% supersaturation. Aerosol size distributions showed very little change and the submicron organic mass fraction increased by less than 15% throughout the experiment; as such, neither of these typical metrics can explain the observed reduction in hygroscopicity. Predictions of the hygroscopicity parameter that make the common assumption that all particles have the same bulk organic volume fractions lead to overpredictions of CCN concentrations by 25% in these experiments. Importantly, key changes in sea spray aerosol mixing state that ultimately influenced CCN activity were driven by bacteria-mediated alterations to the organic composition of seawater.

Daniel, P, Jeremiah J. M, Emily K, Mingxun W, Margot E. W, Eric E. A, Lihini I. A, Pieter C. D.  2019.  Non-Targeted Metabolomics Enables the Prioritization and Tracking of Anthropogenic Pollutants in Coastal Seawater.   10.26434/chemrxiv.9817133.v1   Abstract

Anthropogenic pollutants inundate marine ecosystems as human population growth and urbanization rapidly increase along the coast. Our analytical methods are typically aimed at measuring and monitoring a restricted number of compounds. To prioritize coastal anthropogenic impacts in a comprehensive fashion, we applied large-scale non-targeted liquid chromatography tandem mass spectrometry. We integrated an advanced data analysis pipeline that allows scalable comparison of chemotypes between samples in addition to expanded compound annotation using molecular networking. Using this workflow, we explored the chemical impacts of a major rain event in January 2018 in coastal San Diego, USA. We observed the seawater chemotype shift significantly after the rain event. Molecular drivers of this shift could be attributed to multiple anthropogenic compounds, such as pesticides, cleaning products, drugs and chemical additives that could be connected to potential point sources. Expanding the search of identified xenobiotics to other public tandem mass spectrometry datasets, we could further contextualize their origin and show their potential importance in other ecosystems. Ultimately, the mass spectrometry and data analysis pipeline applied here offer a scalable framework for future molecular mapping and monitoring of marine ecosystems, which we hope will contribute to a more deliberate assessment of how chemical pollution impacts marine environments.

Eglinton, TI, Aluwihare LI, Bauer JE, Druffel ERM, McNichol AP.  1996.  Gas chromatographic isolation of individual compounds from complex matrices for radiocarbon dating. Analytical Chemistry. 68:904-912.   10.1021/ac9508513   AbstractWebsite

This paper describes the application of a novel, practical approach for isolation of individual compounds from complex organic matrices for natural abundance radiocarbon measurement. This is achieved through the use of automated preparative capillary gas chromatography (PCGC) to separate and recover sufficient quantities of individual target compounds for C-14 analysis by accelerator mass spectrometry (AMS). We developed and tested this approach using a suite of samples (plant lipids, petroleums) whose ages spanned the C-14 time scale and which contained a variety of compound types (fatty acids, sterols, hydrocarbons), Comparison of individual compound and bulk radiocarbon signatures for the isotopically homogeneous samples studied revealed that Delta(14)C values generally agreed well (+/- 10%). Background contamination was assessed at each stage of the isolation procedure, and incomplete solvent removal prior to combustion was the only significant source of additional carbon, Isotope fractionation was addressed through compound-specific stable carbon isotopic analyses, Fractionation of isotopes during isolation of individual compounds was minimal (< 5 parts per thousand for delta(13)C), provided the entire peak was collected during PCGC, Trapping of partially coeluting peaks did cause errors, and these results highlight the importance of conducting stable carbon isotopic measurements of each trapped compound in concert with AMS for reliable radiocarbon measurements, The addition of carbon accompanying derivatization of functionalized compounds (e.g., fatty acids and sterols) prior to chromatographic separation represents a further source of potential error, This contribution can be removed using a simple isotopic mass balance approach, Based on these preliminary results, the PCGC-based approach holds promise for accurately determining C-14 ages on compounds specific to a given source within complex, heterogeneous samples.

Gaston, CJ, Furutani H, Guazzotti SA, Coffee KR, Bates TS, Quinn PK, Aluwihare LI, Mitchell BG, Prather KA.  2011.  Unique ocean-derived particles serve as a proxy for changes in ocean chemistry. Journal of Geophysical Research-Atmospheres. 116   10.1029/2010jd015289   AbstractWebsite

Oceans represent a significant natural source of gases and particles to the atmosphere. Relative to gas phase compounds, less is known regarding the influence of changes in biological activity in the ocean on the chemistry of sea spray aerosols produced in marine environments. To gain insight into the influence of ocean biology and chemistry on atmospheric aerosol chemistry, simultaneous real-time measurements were made of atmospheric aerosol size and chemical mixing-state, gas phase dimethyl sulfide (DMS), as well as seawater DMS and chlorophyll a. In three different marine environments with elevated chlorophyll a and DMS, unique Mg particles were detected containing Mg(2+), Ca(2+), K(+), and organic carbon. These particles were segregated from sea salt particles highlighting that two subpopulations within the sea spray were being ejected from the ocean. Strong temporal correlations were observed between these unique ocean-derived particles and freshly emitted sea salt particles (R(2) = 0.86), particularly as wind speed increased to at least 10 m/s, and atmospheric DMS (R(2) = 0.76). Time series correlations between ocean measurements and atmospheric aerosol chemistry suggest that chlorophyll a and DMS serve as indicators of changes in the chemistry of the ocean, most likely an increase in organic material, which is directly reflected in the single particle mixing-state. This is the first time such real-time correlations are shown between ocean chemistry and atmospheric aerosol mixing-state. The reasons behind these observed changes in aerosol chemistry are critical for understanding the heterogeneous reactivity, water uptake, and cloud forming potential of sea spray aerosols.

Goldberg, SJ, Ball GI, Allen BC, Schladow SG, Simpson AJ, Masoom H, Soong R, Graven HD, Aluwihare LI.  2015.  Refractory dissolved organic nitrogen accumulation in high-elevation lakes. Nature Communications. 6   10.1038/ncomms7347   AbstractWebsite

The role of dissolved organic matter (DOM) as either a sink for inorganic nutrients or an additional nutrient source is an often-neglected component of nutrient budgets in aquatic environments. Here, we examined the role of DOM in reactive nitrogen (N) storage in Sierra Nevada (California, USA) lakes where atmospheric deposition of N has shifted the lakes toward seasonal phosphorus (P)-limitation. Nuclear magnetic resonance (NMR) spectroscopy and isotope analyses performed on DOM isolated from Lake Tahoe reveal the accumulation of refractory proteinaceous material with a 100-200-year residence time. In contrast, smaller lakes in the same watershed contain DOM with typical terrestrial characteristics, indicating that proteins in Lake Tahoe are autochthonously produced. These data support the role of DOM as a possible sink for reactive N in these lake ecosystems and identify a potential role for DOM in affecting the inorganic nutrient stoichiometry of these environments.

Hansman, RL, Thurber AR, Levin LA, Aluwihare LI.  2017.  Methane fates in the benthos and water column at cold seep sites along the continental margin of Central and North America. Deep-Sea Research Part I-Oceanographic Research Papers. 120:122-131.   10.1016/j.dsr.2016.12.016   AbstractWebsite

The potential influence of methane seeps on carbon cycling is a key question for global assessments, but the study of carbon cycling in surface sediments and the water column of cold seep environments is complicated by the high temporal and spatial variability of fluid and gas fluxes at these sites. In this study we directly examined carbon sources supporting benthic and planktonic food webs at venting methane seeps using isotopic and molecular approaches that integrate this variability. At four seep environments located along North and Central America, microorganisms from two size fractions were collected over several days from 2800 to 90501 of seawater to provide a time-integrated measure of key microbial groups and the carbon sources supporting the overall planktonic microbial community. In addition to water column measurements, the extent of seafloor methane release was estimated at two of the sites by examining the stable carbon isotopic signature (delta C-13) of benthic metazoan infauna. This signature reveals carbon sources fueling the base of the food chain and thus provides a metric that represents a time-integrated view of the dominant microbial processes within the sediment. The stable carbon isotopic composition of microbial DNA (delta C-13-DNA), which had values between -17.0 and -19.5%(0), indicated that bulk planktonic microbial production was not ultimately linked to methane or other C-13-depleted seep-derived carbon sources. Instead these data support the importance of organic carbon derived from either photo- or chemoautotrophic CO2 fixation to the planktonic food web. Results of qPCR of microbial DNA sequences coding for a subunit of the particulate methane monooxygenase gene (pmoA) showed that only a small percentage of the planktonic microbial community were potential methane oxidizers possessing pmoA (< 5% of 16S rRNA gene copies). There was an overall decrease of C-13-depleted carbon fueling the benthic metazoan community from 3 to 5 cm below the seafloor to the sediment surface, reflecting limited use of isotopically depleted carbon at the sediment surface. Rare methane emission as indicated by limited aerobic methane oxidation acts to corroborate our findings for the planktonic microbial community.

Hansman, RL, Griffin S, Watson JT, Druffel ERM, Ingalls AE, Pearson A, Aluwihare LI.  2009.  The radiocarbon signature of microorganisms in the mesopelagic ocean. Proceedings of the National Academy of Sciences of the United States of America. 106:6513-6518.   10.1073/pnas.0810871106   AbstractWebsite

Several lines of evidence indicate that microorganisms in the meso-and bathypelagic ocean are metabolically active and respiring carbon. In addition, growing evidence suggests that archaea are fixing inorganic carbon in this environment. However, direct quantification of the contribution from deep ocean carbon sources to community production in the dark ocean remains a challenge. In this study, carbon flow through the microbial community at 2 depths in the mesopelagic zone of the North Pacific Subtropical Gyre was examined by exploiting the unique radiocarbon signatures (Delta(14)C) of the 3 major carbon sources in this environment. The radiocarbon content of nucleic acids, a biomarker for viable cells, isolated from size-fractionated particles (0.2-0.5 mu m and >0.5 mu m) showed the direct incorporation of carbon delivered by rapidly sinking particles. Most significantly, at the 2 mesopelagic depths examined (670 m and 915 m), carbon derived from in situ autotrophic fixation supported a significant fraction of the free-living microbial community (0.2-0.5 mu m size fraction), but the contribution of chemoautotrophy varied markedly between the 2 depths. Results further showed that utilization of the ocean's largest reduced carbon reservoir, (14)C-depleted, dissolved organic carbon, was negligible in this environment. This isotopic portrait of carbon assimilation by the in situ, free-living microbial community, integrated over > 50,000 L of seawater, implies that recent, photosynthetic carbon is not always the major carbon source supporting microbial community production in the mesopelagic realm.

Haskell, WZ, Prokopenko MG, Hammond DE, Stanley RHR, Berelson WM, Baronas JJ, Fleming JC, Aluwihare L.  2016.  An organic carbon budget for coastal Southern California determined by estimates of vertical nutrient flux, net community production and export. Deep-Sea Research Part I-Oceanographic Research Papers. 116:49-76.   10.1016/j.dsr.2016.07.003   AbstractWebsite

Organic carbon export and burial in coastal upwelling regions is an important mechanism for oceanic uptake of atmospheric CO2. In order to understand how these complex systems will respond to future climate forcing, further studies of nutrient input, biological production and export are needed. Using a Be-7-based approach, we produced an 18-month record of upwelling velocity estimates at the San Pedro Ocean Time-series (SPOT), Southern California Bight. These upwelling rates and vertical nutrient distributions have been combined to make estimates of potential new production (PNP), which are compared to estimates of net community oxygen production (NOP) made using a one-dimensional, two-box non-steady state model of euphotic zone biological oxygen supersaturation. NOP agrees within uncertainty with PNP, suggesting that upwelling is the dominant mechanism for supplying the ecosystem with new nutrients in the spring season, but negligible in the fall and winter. Combining this data set with estimates of sinking particulate organic carbon (POC) flux from water column Th-234:U-238 disequilibrium and sediment trap deployments, and an estimate of the ratio of dissolved organic carbon (DOC):POC consumption rates, we construct a simple box model of organic carbon in the upper 200 m of our study site. This box model (with uncertainties of +/- 50%) suggests that in spring, 28% of net production leaves the euphotic zone as DOC, of this, similar to 12% as horizontal export and 16% via downward mixing. The remaining similar to 72% of net organic carbon export exits as sinking POC, with only 10% of euphotic zone export reaching 200 m. We find the metabolic requirement for the local heterotrophic community below the euphotic zone, but above 200 m, is similar to 105 +/- 50 mmol C m(-2) d(-1), or similar to 80% of net euphotic zone production in spring. (C) 2016 Elsevier Ltd. All rights reserved.

Ingalls, AE, Shah SR, Hansman RL, Aluwihare LI, Santos GM, Druffel ERM, Pearson A.  2006.  Quantifying archaeal community autotrophy in the mesopelagic ocean using natural radiocarbon. Proceedings of the National Academy of Sciences of the United States of America. 103:6442-6447.   10.1073/pnas.0510157103   AbstractWebsite

An ammonia-oxidizing, carbon-fixing archaeon, Candidatus "Nitrosopumilus maritimus," recently was isolated from a salt-water aquarium, definitively confirming that chemoautotrophy exists among the marine archaea. However, in other incubation studies, pelagic archaea also were capable of using organic carbon. It has remained unknown what fraction of the total marine archaeal community is autotrophic in situ. If archaea live primarily as autotrophs in the natural environment, a large ammonia-oxidizing population would play a significant role in marine nitrification. Here we use the natural distribution of radiocarbon in archaeal membrane lipids to quantify the bulk carbon metabolism of archaea at two depths in the subtropical North Pacific gyre. Our compound-specific radiocarbon data show that the archaea in surface waters incorporate modern carbon into their membrane lipids, and archaea at 670 m incorporate carbon that is slightly more isotopically enriched than inorganic carbon at the same depth. An isotopic mass balance model shows that the dominant metabolism at depth indeed is autotrophy (83%), whereas heterotrophic consumption of modern organic carbon accounts for the remainder of archaeal biomass. These results reflect the in situ production of the total community that produces tetraether lipids and are not subject to biases associated with incubation and/or culture experiments. The data suggest either that the marine archaeal community includes both autotrophs and heterotrophs or is a single population with a uniformly mixotrophic metabolism. The metabolic and phylogenetic diversity of the marine archaea warrants further exploration; these organisms may play a major role in the marine cycles of nitrogen and carbon.

Kharbush, JJ, Allen AE, Moustafa A, Dorrestein PC, Aluwihare LI.  2016.  Intact polar diacylglycerol biomarker lipids isolated from suspended particulate organic matter accumulating in an ultraoligotrophic water column. Organic Geochemistry. 100:29-41.   10.1016/j.orggeochem.2016.07.008   Abstract

Intact polar diacylglycerols (IP-DAGs) are essential components of cell membranes. Because they are structurally diverse and hypothesized to represent primarily living cells, they are potential molecular markers for a recent contribution by microbial communities to various carbon reservoirs. This study employed a novel molecular networking approach to investigate the evolution of IP-DAG structural diversity with depth in an ultraoligotrophic environment of the western South Pacific Ocean to test the hypothesis that particle transport to depth is rapid enough to preserve the IP-DAG biomarker signature of the photosynthetic community. IP-DAG profiles of several cultured cyanobacteria and photosynthetic picoeukaryotes were used as templates for constructing molecular networks to compare and interpret IP-DAG signatures of suspended particles isolated from a water column depth profile. Analysis of corresponding genetic community composition data for the same field samples was used to connect IP-DAG structures with their likely biological sources. Our data show that, although most IP-DAG classes associated with photosynthetic organisms were not observed below the euphotic zone, several other IP-DAG classes in deep samples might provide interesting targets for future studies seeking to examine the in situ contribution of deep sea microbes to suspended particulate organic matter (POM). Overall, the results represent the deepest water column IP-DAG dataset to date and demonstrate the utility of molecular networking for analyzing and visualizing complex environmental datasets.

Kharbush, JJ, Ugalde JA, Hogle SL, Allen EE, Aluwihare LI.  2013.  Composite bacterial hopanoids and their microbial producers across oxygen gradients in the water column of the California Current. Applied and Environmental Microbiology.   10.1128/aem.02367-13   AbstractWebsite

Hopanoids are pentacyclic triterpenoid lipids produced by many prokaryotes as cell membrane components. The structural variations of composite hopanoids, or bacteriohopanepolyols (BHPs), produced by various bacterial genera makes them potentially useful molecular biomarkers of bacterial communities and metabolic processes, in both modern and ancient environments. Building on previous work suggesting that organisms in low-oxygen environments are important contributors to BHP production in the marine water column and that there may be physiological roles for BHPs specific to these environments, this study investigated the relationship between trends in BHP structural diversity and abundance and the genetic diversity of BHP producers for the first time in a low-oxygen environment of the Eastern Tropical North Pacific. Amplification of the hopanoid biosynthesis gene, squalene hopene cyclase (sqhC), indicated far greater genetic diversity than would be predicted by examining BHP structural diversity alone, and that greater sqhC genetic diversity exists in the marine environment than is represented by cultured representatives and most marine metagenomes. In addition, the genetic relationships in this dataset suggest microaerophilic environments as potential “hot spots” of BHP production. Finally, structural analysis of BHPs showed that an isomer of the commonly-observed BHP bacteriohopanetetrol (BHT) may be linked to a producer that is more abundant in low-oxygen environments. Results of this study increase the known diversity of BHP producers and provide a detailed phylogeny with implications for the role of hopanoids in modern bacteria as well as the evolutionary history of hopanoid biosynthesis, both of which are important considerations for future interpretations of the marine sedimentary record.

Kharbush, JJ, Kejriwal K, Aluwihare LI.  2016.  Distribution and abundance of hopanoid producers in low-oxygen environments of the Eastern Pacific Ocean. Microbial Ecology. 71:401-408.   10.1007/s00248-015-0671-y   AbstractWebsite

Hopanoids are bacterial membrane lipid biomarker molecules that feature prominently in the molecular fossil record. In the modern marine water column, recent reports implicate bacteria inhabiting low-oxygen environments as important sources of hopanoids to marine sediments. However, the preliminary biogeography reported by recent studies and the environmental conditions governing such distributions can only be confirmed when the numerical abundance of these organisms is known with more certainty. In this study, we employ two different approaches to examine the quantitative significance of phylogenetically distinct hopanoid producers in low-oxygen environments. First, we develop a novel quantitative PCR (qPCR) assay for the squalene hopene cyclase (sqhC) gene, targeting a subset of hopanoid producers previously identified to be important in the eastern North Pacific Ocean. The results represent the first quantitative gene abundance data of any kind for hopanoid producers in the marine water column and show that these putative alphaproteobacterial hopanoid producers are rare, comprising at most 0.2 % of the total bacterial community in our samples. Second, a complementary analysis of existing low-oxygen metagenomic datasets further examined the generality of the qPCR observation. We find that the dominant sqhC sequences in these metagenomic datasets are associated with phyla such as Nitrospinae rather than Proteobacteria, consistent with the qPCR finding that alphaproteobacterial hopanoid producers are not very abundant in low-oxygen environments. In fact, positive correlations between sqhC gene abundance and environmental parameters in these samples identify nitrite availability as a potentially important factor in the ecology of hopanoid producers that dominate low-oxygen environments.

Kharbush, JJ, Thompson LR, Haroon MF, Knight R, Aluwihare LI.  2018.  Hopanoid-producing bacteria in the Red Sea include the major marine nitrite oxidizers. Fems Microbiology Ecology. 94   10.1093/femsec/fiy063   AbstractWebsite

Hopanoids, including the extended side chain-containing bacteriohopanepolyols, are bacterial lipids found abundantly in the geological record and across Earth's surface environments. However, the physiological roles of this biomarker remain uncertain, limiting interpretation of their presence in current and past environments. Recent work investigating the diversity and distribution of hopanoid producers in the marine environment implicated low-oxygen regions as important loci of hopanoid production, and data from marine oxygen minimum zones suggested that the dominant hopanoid producers in these environments are nitrite-utilizing organisms, revealing a potential connection between hopanoid production and the marine nitrogen cycle. Here, we use metagenomic data from the Red Sea to investigate the ecology of hopanoid producers in an environmental setting that is biogeochemically distinct from those investigated previously. The distributions of hopanoid production and nitrite oxidation genes in the Red Sea are closely correlated, and the majority of hopanoid producers are taxonomically affiliated with the major marine nitrite oxidizers, Nitrospinae and Nitrospirae. These results suggest that the relationship between hopanoid production and nitrite oxidation is conserved across varying biogeochemical conditions in dark ocean microbial ecosystems.

Kumar, A, Borgen M, Aluwihare LI, Fenical W.  2017.  Ozone-activated halogenation of mono- and dimethylbipyrrole in seawater. Environmental Science & Technology. 51:589-595.   10.1021/acs.est.6b03601   AbstractWebsite

Polyhalogenated N-methylbipyrroles of two different structure classes have been detected worldwide in over 100 environmental samples including seawater, bird eggs, fish, dolphin blubber, and in the breast milk of humans that consume seafood. These molecules are concentrated in the fatty tissues in comparable abundance to some of the most important anthropogenic contaminants, such as the halogenated flame-retardants and pesticides. Although the origin of these compounds is still unknown, we present evidence that the production of these materials can involve the direct ozone activated seawater halogenation of N-methylbipyrrole precursors. This observation shows that environmental polyhalogenated bipyrroles can be produced via an abiotic process, and implies that the ozone activated halogenation of a variety of natural and anthropogenic seawater organics may be a significant process occurring in surface ocean waters.