Dissolved Free Amino Acids Research Articles

High-throughput determination of dissolved free amino acids in unconcentrated freshwater by ion-pairing liquid chromatography and mass spectrometry

We developed a procedure for the direct determination of dissolved free amino acids (DFAAs) in freshwater samples employing ion-pairing liquid chromatography and mass spectrometry. Our approach allowed accurate quantification of subnanomolar concentrations of DFAAs without prior concentration, derivatization or sample clean-up steps, achieving a throughput of three samples per hour. DFAAs were separated on a C-18 resin using tridecafluoroheptanoic acid as an ion-pairing agent controlling the overall retention. The relative standard deviation of DFAA measurements was <10% in samples from the mesotrophic Lake Zurich (Switzerland), and across concentrations of 0.5–500nM. Recoveries of DFAAs ranged from 94 to 102% within the range of 0.2–10nM. The limits of quantification for individual DFAAs varied between 50pM to 2nM (median, 0.5nM). The new method was employed to compare the spatial variability of DFAA concentrations in samples obtained by two devices. Epilimnetic samples of different size (ml, l) were collected at various spatial scales (cm, m, km) with a traditional 5l Friedinger sampler and with a custom-made multi-syringe sampling apparatus. Concentrations of total DFAAs ranged from 30 to 330nM. Alanine, serine, glutamic acid, arginine and glycine constituted 65% of the total pool, while methionine and tryptophan occurred at sub-nM concentrations only. Concentrations of individual DFAAs varied spatially over 2 orders of magnitude. Their spatial distribution was positively skewed, as characterized by rare peaks, most strongly so for glutamate, glycine and asparagine. The composition of DFAAs significantly differed at all examined spatial scales, and this could be mainly attributed to alanine, aspartic acid, and glycine. Our new method equals or outperforms existing ones in terms of sensitivity and reproducibility, while its procedural simplicity renders it superior for the high-throughput analysis of freshwater samples.

Analysis of meat flavor compounds in pedigree and two-strain Yangzhou geese

In this study, we evaluated the meat flavor compounds of Yangzhou geese, including one group of pedigree strain (AA group) and 4 groups of 2-strain crossbreds (KA, KB, CA, and SA). Each group consisted of 100 geese comprised of 5 replicates of 10 males and 10 females each. Inosine 5′-monophosphate (IMP), amino acid (AA), and fatty acid (FA) levels in breast and thigh muscle were determined. Results showed that AA group had the highest levels of total amino acid (TAA) and dissolved free amino acids (DFAA) in breast muscle and of polyunsaturated fatty acids (PUFA) in thigh muscle (P < 0.05). In SA group, the levels of C17:1, C22:0, C22:1, C20:4, and C24:1 in breast muscle were significantly higher in SA than in other groups (P < 0.05). KB group had the lowest glycine levels in breast muscle (P < 0.05) while MUFA levels were significantly higher in KB than in other groups (P < 0.05). In KA, the levels of C18:3 in breast muscle in were higher than in CA and KB (P < 0.05). CA had relevant higher IMPc levels in breast muscle than SA (P < 0.05) and other groups (P > 0.05); however, no significant differences were obtained in thigh muscle (P > 0.05). In conclusion, Yangzhou AA goose has high levels of meat flavor compounds than its crossbreeds. Future efforts should focus on assessing meat flavor through measurement of sensory characteristics of Yangzhou geese.

Dissolved organic nitrogen bioavailability indicated by amino acids during a diatom to dinoflagellate bloom succession in the Changjiang River estuary and its adjacent shelf

This study investigates the bioavailability and potential nutritional role of dissolved organic nitrogen (DON) in the development and progression of a large-scale dinoflagellate bloom that occurred in the Changjiang River estuary and its adjacent shelf (CJREAS) in the East China Sea (ECS). Three cruises were conducted during the late spring and early summer following the bloom progression, i.e., the termination phase of the diatom bloom, and the initiation/growth and peak phases of the dinoflagellate bloom. Concentrations of dissolved organic carbon (DOC), DON, dissolved inorganic nitrogen (DIN), dissolved free amino acids (DFAA) and dissolved combined amino acids (DCAA) were measured. The degradation index (DI), based on the relative abundance of total dissolved amino acids (TDAA) and amino acids yields was applied to indicate the diagenetic alteration of dissolved organic matter (DOM). During the succession from a diatom to dinoflagellate bloom, a decrease in DON co-occurred with a decrease in DIN. Concentrations of DFAA and DCAA also clearly decreased. The yields of DFAA and the DCAA and DI values decreased concomitantly with an increase in the DOC/DON, indicating that bioavailability of DON was relatively higher during the end stage of the diatom bloom than during the peak of the dinoflagellate bloom. These results indicated that DON may provide a significant portion of the total N demand throughout the duration of the large-scale dinoflagellate bloom under conditions of low DIN availability in the CJREAS.

Nitrogen dynamics in the Irish Sea and adjacent shelf waters: An exploration of dissolved organic nitrogen

Relatively little is known about dissolved organic nitrogen (DON) in the marine environment because research has historically focused on dissolved inorganic nitrogen (DIN). In this study we combine measurements of dissolved organic matter (DOM), DIN, particulate organic nitrogen (PON), dissolved inorganic phosphorus (DIP) and silicon (DIS), with temperature and salinity data from the western shelf region of the UK and Ireland, and with inorganic and organic nitrogen (N) data from the western Irish Sea to develop an understanding of N dynamics in the Irish Sea and adjacent shelf waters, and investigate the role of DON in the nitrogen budget of the seasonally stratifying western Irish Sea. In January 2013, the sampling area was divided by density fronts into 4 regions of distinct oceanography and homogeneous chemistry. DON concentrations accounted for 25.3 ± 1.8% of total dissolved N (TDN) across all regions. DOM concentrations generally decreased from the freshwater influenced water of Liverpool Bay to the oceanic waters of the Celtic Sea and Malin Shelf. Urea and dissolved free amino acids (DFAA) together made up 27.3 ± 3.1% of DON. Estimated concentrations in the rivers discharging into Liverpool Bay were 8.0 and 2.1 μmol N L−1 respectively: at the high end of reported riverine concentrations. Oceanic nutrient inputs to the Irish Sea only have a small influence on N concentrations. Riverine N inputs to the Irish Sea are substantial but are likely removed by natural N cycling processes. In the western Irish Sea, DON and PON concentrations reached maxima and minima in midsummer and early spring respectively. DIN followed the opposite trend. DON accounted for 38% of the yearly internal N cycling and we estimated that as much as 1.4 ± 1.2 μmol N L−1 of labile DON was available as an N source at the start of the spring bloom. Our study supports the view that DON plays an important role in N cycling in temperate shelf and coastal seas and should be included more often in biogeochemical measurements if we are to have a complete understanding of N dynamics in a changing world.

Temporal dynamics and depth variations of dissolved free amino acids and polyamines in coastal seawater determined by high-performance liquid chromatography

Short-chained aliphatic polyamines (PAs) are a class of labile dissolved organic nitrogen (DON) that has biogeochemical similarities to dissolved free amino acids (DFAAs). Here we investigated the relative contributions of DFAAs and PAs to the total DON pool and their diurnal dynamics at different depths at the Gray's Reef National Marine Sanctuary (GRNMS) in the spring and fall of 2011. A high-performance liquid chromatography (HPLC) method that uses pre-column fluorometric derivatization with o-phthaldialdehyde, ethanethiol, and 9-fluorenylmethyl chloroformate was optimized to measure 20 DFAAs and 5 PAs in seawater simultaneously. The concentrations of DFAAs and PAs varied over 5-fold during individual diurnal cycles and between seasons; and concentrations of the former (tens to hundreds nM) were typically at least one order of magnitude higher than the latter (a few nM). An exception was noted in fall surface water samples when the total PAs reached 159nM and the ratio of PAs to DFAAs was about 2:3. Compositions of individual DFAAs and PAs also exhibited temporal dynamics, with glycine and spermidine consistently the most abundant compound in each pool, respectively. DFAA concentrations appeared to track chlorophyll a, whereas, total PA concentrations were strongly correlated with bacterial cell abundance. Our results indicate that, at least occasionally, PAs may serve as an important DON pool at the GRNMS. This view is in accordance with recent molecular data but contrasts to measurements made in some other marine environments.

Concentrations and distributions of amino acids in black and white smoker fluids at temperatures over 200 °C

The distribution of amino acids in seafloor hydrothermal systems was investigated through the determination of the concentrations of total hydrolysable amino acids (THAAs), dissolved free amino acids (DFAAs) and cell density in>200°C black and white smoker fluids sampled from the Mariana Trough. THAA concentrations of>10μM were detected in the black and white smoker fluids, which are higher than those of low temperature (< 53°C) fluids and ambient seawater (< 1μM). 1.4×104–8.6×105cell/ml of microbe was detected from low temperature hydrothermal fluids (< 100°C) and ambient seawater. The concentration of THAAs increased with increasing temperature, although the cell density decreased in high temperature fluid (> 150°C). The bioactivity would be restricted under the high temperature condition.Levels of DFAAs (< 0.7μM) were very low, suggesting that the amino acids existed mainly as polymers in these hydrothermal fluids. The amino acid polymers plausibly derive from biological protein and dissolve during the reaction of hydrothermal fluids along flow paths around the hydrothermal vents.Amino acids are considered to be unstable under hydrothermal condition (> 200°C). However, labile amino acids (e.g., Asp and Ser) were abundant in high temperature fluids. These amino acids would be protected by reaction with inorganic compounds. The behavior of the amino acids derived from organisms around hydrothermal vents would be constrained more by abiotic physico-chemical reactions than biological activities in hydrothermal systems.

Characteristics and transformations of dissolved organic nitrogen in municipal biological nitrogen removal wastewater treatment plants

Dissolved organic nitrogen (DON) represents most of the dissolved nitrogen in the effluent of biological nitrogen removal (BNR) wastewater treatment plants (WWTPs). The characteristics of wastewater-derived DON in two different WWTPs were investigated by several different methods. The major removals of DON and biodegradable dissolved organic nitrogen (BDON) along the treatment train were observed in the anaerobic process. Dissolved combined amino acids (DCAA) and dissolved free amino acids (DFAA) in the effluent accounted approximately for less than 4% and 1% of the effluent DON, respectively. Approximately half of wastewater-derived DON was capable of passing through a 1 kDa ultrafilter, and low MW DON cannot effectively be removed by BNR processes. More than 80% of effluent DON was composed of hydrophilic compounds, which stimulate algal growth. The study provided important information for future upgrading of WWTPs or the selection of DON removal systems to meet more demanding nitrogen discharge limits.

Olfactory Homing of Chum Salmon to Stable Compositions of Amino Acids in Natal Stream Water

Many attempts have been made to identify natal stream odors for salmon olfactory homing. It has recently been hypothesized that odors are dissolved free amino acids; however, it is unknown whether these odors change on a seasonal or annual basis. We analyzed dissolved free amino acid (DFAA) concentration and composition of water from the Teshio River in Hokkaido, Japan, where chum salmon (Oncorhynchus keta) returned for spawning, during juvenile downstream migration in spring and adult upstream migration in autumn with a 4-year difference. Among the 19 amino acids found in the Teshio River water, DFAA concentrations fluctuated largely, but 5-7 stable DFAA compositions (mole %) were found between the spring and autumn samples over a 4-year span. Two kinds of artificial stream water (ASW) were prepared using the same DFAA concentration in the Teshio River during the time of juvenile imprinting in spring (jASW) and adult homing in autumn (aASW), after a 4-year period. In behavioral experiments of upstream selective movement in a 2choice test tank, 4-year-old mature male chum salmon captured in the Teshio River showed significant preference for either jASW or aASW when compared to control water, but did not show any preference with respect to jASW or aASW. In electro-olfactogram experiments, adults were able to discriminate between jASW and aASW. Our findings demonstrate that the long-term stability of the DFAA compositions in natal streams may be crucial for olfactory homing in chum salmon.

Uptake of dissolved free amino acids by four cold-water coral species from the Mediterranean Sea

Dissolved organic matter, which contains many compounds such as lipids, sugars and amino acids, is an important source of carbon and nitrogen for several symbiotic and asymbiotic tropical coral species. However, there is still no information on its possible uptake by cold-water coral species. In this study, we demonstrated that dissolved organic matter, in the form of dissolved free amino acids (DFAA), is actively absorbed by four cold-water coral species from the Mediterranean Sea. Although the uptake rates observed with 3µM DFAA concentration were one order of magnitude lower than those observed in tropical species, they corresponded to 12–50% of the daily excreted-nitrogen, and 16–89% of the daily respired-carbon of the cold-water corals. Consequently, DFAA, even at in situ concentrations lower than those tested in this study, can supply a significant amount of carbon and nitrogen to the corals, especially during periods when particulate food is scarce.

Phytoplankton species‐specific release of dissolved free amino acids and their selective consumption by bacteria

Despite representing only a small fraction of the ocean's dissolved organic matter pool, dissolved free amino acids (DFAA) have high turnover rates and are major nitrogen and carbon sources for bacterioplankton. Both phytoplankton and bacterioplankton assimilate and release DFAA, but their consumption and production are difficult to quantify in nature due to their short residence times (min) as dissolved monomers. We segregated DFAA production by phytoplankton and bacterial consumption by measuring individual DFAA concentrations in four axenic phytoplankton cultures during the exponential growth phase, and also after 4 d incubations in the presence of a natural bacterioplankton community. The amounts and composition of the DFAA pool varied widely among phytoplankton species. The proportion of dissolved organic carbon attributed to DFAA varied among cultures. The picoeukaryotic prasinophyte, Micromonas pusilla, released higher amounts of DFAA than the other species tested (diatoms and dinoflagellate), especially alanine, which has been reported as the dominant individual DFAA in some oligotrophic environments. Community structure of heterotrophic prokaryotes responded to differences in the quality of organic matter released among microalgal species, with Roseobacter‐related bacteria responding strongly to exudate composition. Our results demonstrate the specificity of DFAA extracellular release among several algal species and their preferential uptake by members of bacterial communities.

Improvements in the Separation Capabilities of Sequential Injection Chromatography: Determination of Intracellular Dissolved Free Amino Acid Profiles in Three Taxonomic Groups of Microalgae

Dissolved free amino acids (DFAA) in intracellular extracts of marine microalgae can be determined by sequential injection chromatography (SIC). This technique uses portable, low-cost instrumentation but its applications have been limited to short monolithic columns because of components not resistant to high pressures. To develop a SIC method for determination of DFAA by exploring an instrument modified to handle pressures of 1000 psi. The method was based on pre-column derivatisation of the amino acids with o-phthalaldehyde and 2-mercaptoethanol in borate buffer (pH 9.4), separation and fluorimetric detection (λ(excitation)= 340 and λ(emission)= 450 nm). Separation was achieved by stepwise gradient elution using six mobile phases. The first elution step used a mobile phase composed of methanol:tetrahydrofuran:10 mm phosphate buffer (pH 7.2) at a volumetric ratio of 8:1:91. Additional elution steps used mobile phases containing methanol and 10 mM phosphate buffer at ratios of 17.5:82.5, 25:75, 35:65, 50:50 and 65:35. Nineteen chromatographic peaks were observed in a mixture of 20 amino acids. The only complete co-elution was between tryptophan and methionine. Detection limits varied from 0.10 µm for isoleucine to 1.5 µm for lysine. Recoveries from spiked extracts were between 84 and 131%. Resolutions of the amino acid pairs glutamine and histidine, valine and phenylalanine, and isoleucine and leucine were 1.5, 0.75 and 1.3, respectively. The proposed method found different profiles of DFAA among the three species of algae, suggesting its adequacy for metabolic studies.

Growth and release of extracellular organic compounds by benthic diatoms depend on interactions with bacteria

Phototrophic epilithic biofilms harbour a distinct assemblage of heterotrophic bacteria, cyanobacteria and photoautotrophic algae. Secretion of extracellular polymeric substances (EPS) by these organisms and the physicochemical properties of the EPS are important factors for the development of the biofilms. We have isolated representative diatom and bacteria strains from epilithic biofilms of Lake Constance. By pairwise co-cultivating these strains we found that diatom growth and EPS secretion by diatoms may depend on the presence of individual bacteria. Similar results were obtained after addition of spent bacterial medium to diatom cultures, suggesting that soluble substances from bacteria have an impact on diatom physiology. While searching for putative bacterial signal substances, we found that concentrations of various dissolved free amino acids (DFAA) within the diatom cultures changed drastically during co-cultivation with bacteria. Further, the secretion of extracellular carbohydrates and proteins can be influenced by bacteria or their extracellular substances. We have performed mass spectrometric peptide mapping to identify proteins which are secreted when co-cultivating the diatom Phaeodactylum tricornutum Bohlin and Escherichia coli. The identified proteins are possibly involved in signalling, extracellular carbohydrate modification and uptake, protein and amino acid modification, and cell/cell aggregation of diatom and bacteria strains. Our data indicate that diatom-bacteria biofilms might be regulated by a complex network of chemical factors involving EPS, amino acid monomers and other substances. Thus interactions with bacteria can be considered as one of the main factors driving biofilm formation by benthic diatoms.

Short-term and seasonal dynamics of bacterial biomass production and amino acid turnover in the water column of an intertidal ecosystem, the Wadden Sea

Intertidal flat systems exhibit systematic temporal patterns of suspended particulate matter (SPM) and associated properties (particulate organic carbon, chlorophyll) with enhanced con- centrations towards the current velocity maximum (CVM) and reduced concentrations towards slack water. It is unclear how these tidal patterns are reflected in organic matter processing by particle- associated (PA) and free-living (FL) heterotrophic bacteria. Therefore, we studied the abundance of PA and FL bacteria, bacterial biomass production (BP) and turnover of dissolved free amino acids (DFAA) during tidal cycles in a 13 to 16 m deep back-barrier intertidal flat system of the East Frisian Wadden Sea, Germany, in January, April, July and November 2005. Pronounced tidal patterns of microbial parameters were found with distinct differences around the CVM and at slack water. Tidal patterns varied seasonally with higher BP rates and bacterial generation times of 1 to 2 d in April and July. There was a trend of enhanced rates of BP from surface to bottom in April and July with an increasing significance of PA bacteria towards the bottom. Very high depth-integrated daily rates of BP and oxygen undersaturation suggest that this system is net-heterotrophic. A normalization proce- dure revealed systematic tidal patterns of most parameters irrespective of the seasonal situation. Our findings thus demonstrate the presence of tidal patterns of parameters reflecting microbial processes and thus complement previous findings of tidal patterns of SPM-associated properties. The tidally varying microbial activities indicate that the rhythmic tidal currents promote organic matter decom- position by heterotrophic bacteria in intertidal flat systems.

Chemosensory response of marine flagellate towards L- and D- dissolved free amino acids generated during heavy grazing on bacteria.

This study investigated the generation of dissolved free amino acids (DFAA) by the bacterivorous flagellate Rhynchomonas nasuta when feeding on abundant prey. Specifically, it examined whether this flagellate protist exhibits a chemosensory response towards those amino acids. The concentrations of glycine and the L- and D-enantiomers of glutamate, serine, threonine, alanine, and leucine were determined in co-cultures of the flagellate and bacteria. Glycine, L- and D-alanine, and L-serine were found to accumulate under these conditions in amounts that correlated positively with flagellate abundance, suggesting that protists are involved in their generation. Investigations of the chemotactic response of young and old foraging protists to the same amino acids, offered in concentrations similar to those previously generated, showed that glycine elicited the strongest attraction in both age groups. Young protists were strongly attracted to all the assayed amino acids, whereas older protists maintained a high level of attraction only for glycine. These results suggest that glycine generated by protists actively grazing in bacterially enriched patches functions as an infochemical, signaling to foraging protists the presence of available prey in the aquatic environment.

Nitrogen uptake by phytoplankton and bacteria during an induced Phaeocystis pouchetii bloom, measured using size fractionation and flow cytometric sorting

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 61:89-104 (2010) - DOI: https://doi.org/10.3354/ame01414 Nitrogen uptake by phytoplankton and bacteria during an induced Phaeocystis pouchetii bloom, measured using size fractionation and flow cytometric sorting P. B. Bradley1,7,*, M. P. Sanderson1, J. C. Nejstgaard2,3, A. F. Sazhin4, M. E. Frischer5, L. M. Killberg-Thoreson1, P. G. Verity5,†, L. Campbell6, D. A. Bronk1 1Virginia Institute of Marine Science, The College of William & Mary, PO Box 1346, Gloucester Point, Virginia 23062, USA 2Uni Environment, Thormohlensgate 49B, 5020 Bergen, Norway 3Department of Biology, University of Bergen, Thormohlensgate 53A, 5006 Bergen, Norway 4P.P. Shirshov Institute of Oceanology RAS, 36 Nakhimovsky Prospect, Moscow 117997, Russia 5Skidaway Institute of Oceanography, 10 Ocean Science Circle, Savannah, Georgia 31411, USA 6Department of Oceanography, Texas A&M University, College Station, Texas 77843, USA 7Present address: National Oceanic and Atmospheric Administration, 1305 East West Hwy N/MB5, Silver Spring, Maryland 20910, USA *Email: paul.bradley@noaa.gov†Deceased ABSTRACT: Uptake of inorganic and organic nitrogen (N) by phytoplankton and bacteria was investigated during a mesocosm study conducted in Raunefjord, Norway in April 2005. One mesocosm was fertilized with nitrate and phosphate at a ratio of 16:1 and maintained in the light, while one unamended light mesocosm served as a control. Dissolved nutrients, phytoplankton and bacterial biomass, and phytoplankton community composition were monitored throughout the 26 d experiment. Uptake of 15N-labeled ammonium and nitrate, and dual-labeled (15N and 13C) urea and dissolved free amino acids (DFAA) was measured for phytoplankton and bacteria using 2 methods: size fractionation into 0.2–0.8 and >0.8 µm size classes, and flow cytometric sorting based on chlorophyll autofluorescence. Prior to fertilization, dissolved inorganic N concentrations were low and comprised ~5% of total dissolved N. Added nitrate was completely utilized in the amended mesocosm within 10 d, stimulating a large bloom of colonial Phaeocystis pouchetii. Ammonium contributed over half of total measured N uptake by phytoplankton and bacteria in both enclosures, while nitrate and urea each supplied roughly 10 to 25%. Overall, DFAA were a negligible N source to phytoplankton but contributed 11% to total bacterial N uptake. Bacterial uptake represented a significant portion of total uptake of all N forms, especially urea and DFAA. Comparison of the 2 methods for measuring phytoplankton versus bacterial uptake demonstrates how the use of 0.8 µm filters can lead to significant overestimation of phytoplankton N uptake due to the retention of bacterial biomass. KEY WORDS: Nitrogen uptake · Mesocosm experiments · Phaeocystis · Phytoplankton · Bacteria · Flow cytometry Full text in pdf format PreviousCite this article as: Bradley PB, Sanderson MP, Nejstgaard JC, Sazhin AF and others (2010) Nitrogen uptake by phytoplankton and bacteria during an induced Phaeocystis pouchetii bloom, measured using size fractionation and flow cytometric sorting. Aquat Microb Ecol 61:89-104. https://doi.org/10.3354/ame01414Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 61, No. 1. Online publication date: August 30, 2010 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2010 Inter-Research.

Distribution of Roseobacter RCA and SAR11 lineages in the North Sea and characteristics of an abundant RCA isolate

The Roseobacter group and SAR11 clade constitute high proportions of the marine bacterioplankton, but only scarce information exists on the abundance of distinct populations of either lineage. Therefore, we quantified the abundance of the largest cluster of the Roseobacter group, the RCA (Roseobacter clade affiliated) cluster together with the SAR11 clade by quantitative PCR in the southern and eastern North Sea. The RCA cluster constituted up to 15 and 21% of total bacterial 16S ribosomal RNA (rRNA) genes in September 2005 and May 2006, respectively. At a few stations, the RCA cluster exceeded the SAR11 clade, whereas at most stations, SAR11 constituted higher fractions with maxima of 37%. In most samples, only one RCA ribotype was detected. RCA abundance was positively correlated with phaeopigments, chlorophyll, dissolved and particulate organic carbon (POC), turnover rates of dissolved free amino acids (DFAAs), temperature, and negatively correlated with salinity. The SAR11 clade was only correlated with POC (negatively, May) and with DFAA turnover rates (positively, September). An abundant RCA strain, 'Candidatus Planktomarina temperata', was isolated from the southern North Sea. This strain has an identical 16S rRNA gene sequence to the dominant RCA ribotype. Detection of the pufM gene, coding for a subunit of the reaction center of bacteriochlorophyll a, indicates the potential of the isolate for aerobic anoxygenic photosynthesis. Our study shows that a distinct population of the RCA cluster constitutes an abundant bacterioplankton group in a neritic sea of the temperate zone and indicates that this population has an important role during decaying phytoplankton blooms.

Release of dissolved free amino acids from biofilms in stream water

We investigated the origin of dissolved free amino acids (DFAA) in stream water focusing on biofilms in the river bed by means of incubation experiments in the laboratory. Stones were placed in the Toyohira River, Hokkaido, Japan, for 3 months, allowing formation of biofilms, and then incubated for 24 h in the laboratory at stream water temperature. After incubation, the composition and concentrations of DFAA in the incubation solution and total hydrolyzed amino acids (THAA) in biofilms were measured by high-performance liquid chromatography. The amount of chlorophyll a (Chl. a) and the number of bacteria were also measured. The DFAA concentration increased greatly in the biofilm incubation solution, but the DFAA composition (mol %) did not change relative to the inception of incubation, where it was similar to stream water. There was no correlation between the increase in DFAA concentration and the THAA concentration, Chl. a amount, or the number of bacteria in biofilms. These results suggest that biofilms are one of the major sources of DFAA in stream water.

Inorganic and Organic Nitrogen Use by Phytoplankton Along Chesapeake Bay, Measured Using a Flow Cytometric Sorting Approach

Two different approaches to measuring phytoplankton nitrogen (N) use were compared in late summer 2004 along the main axis of Chesapeake Bay. Uptake of 15N-labeled ammonium and nitrate and dual-labeled (15N and 13C) urea and dissolved free amino acids (DFAA) were measured in surface water samples from upper, mid, and lower bay stations. Two distinct methods were used to assess the relative uptake of N substrates by phytoplankton and correct for bacterial artifacts: (1) traditional filtration using Whatman glass fiber (GF/F) filters and (2) flow cytometric (FCM) sorting of chlorophyll-containing cells. The concentration of dissolved inorganic N (DIN) decreased with distance south along the bay, whereas dissolved organic N (DON) concentrations were relatively constant. Absolute N uptake rates measured using the traditional approach exceeded those of FCM-sorted phytoplankton, thereby suggesting the possibility of bacterial “contamination.” Ammonium was the dominant N form used throughout the transect, although FCM-sorted phytoplankton relied more on urea and DFAA as the ratio of DON/DIN increased toward the bay mouth. Overall, ammonium comprised 74 ± 17%, urea 10 ± 9%, DFAA 9 ± 7%, and nitrate 7 ± 12% of total measured N uptake by phytoplankton. Results suggest that bacteria relied primarily on DFAA and ammonium for N nutrition but also used N from urea at a rate similar to that of phytoplankton, whereas bacterial nitrate uptake was insignificant. On average, phytoplankton uptake of ammonium, urea, and DFAA was overestimated by 61%, 53%, and 135%, respectively, as a result of bacterial retention on GF/F filters.

Composition and characterization of colloidal organic matter in the coastal surface waters of Qingdao, China

The composition and characterization of dissolved organic matter (DOM) and colloid organic matter (COM) in the coastal surface waters of Qingdao, China were examined. Water samples were collected in Spring 2008 from the Jiaozhou Bay and its adjacent area and fractionated into total dissolved (< 0.45 μm), colloidal (10 kDa–0.45 μm) and cutoff (< 10 kDa) fractions with the cross-flow ultrafiltration (CFF) system. The excitation–emission-matrix (EEM) spectral properties, the absorbance values at 254 nm ( A 254) and dissolved organic carbon (DOC) of the DOM were observed having the colloidal ratios of 8.7%, 6.2%, and 2.2%, respectively, and exhibiting a decreasing trend. Total dissolved carbohydrates (TCHO) and total hydrolysable amino acids (THAA) comprised 24% of the bulk DOC. Among them, the free monosaccharides (MCHO) and dissolved combined amino acids (DCAA）were the major fractions, followed by polysaccharides (PCHO) and dissolved free amino acids (DFAA). Colloidal TCHO and THAA concentrations were 3.2 µM C and 0.3 µM, respectively. They comprised ∼ 35% of the colloid organic carbon (COC), among which PCHO and DCAA were the dominating fractions. The dissolved carbohydrates and amino acids are mostly concentrated in the cutoff fraction. The A 254 values and DOC concentrations inside the Jiaozhou Bay were apparently higher than those outside, while pH and salinity were slightly lower, which might be due to the influence of terrestrial input and the semi-enclosed bay. The distribution pattern of TCHO was found having basically the same as the distributions of A 254 and DOC.

Concentrations and distributions of dissolved amino acids in fluids from Mid-Atlantic Ridge hydrothermal vents

The concentrations and distributions of dissolved free amino acids (DFAA) were determined in 31 hydrothermal fluid samples from the Logatchev field at 15°N and several hydrothermal vent fields between 4° and 9°S on the Mid-Atlantic ridge, sampled during three research cruises. The samples are derived from black smoker fluids with varying degrees of dilution, with end-member temperatures between 350 and 407°C, as well as of diffuse fluids with temperatures around 10°C. Most of the samples, even the hottest ones, contain significant amounts of DFAA and, with an average of 143 nM, more than in the deep water of the open ocean (below 50 nM, Lee and Bada, 1977). The highest values are found in diffuse fluids and the lowest in the hot fluids, but there are also some high-T fluids with high, as well as diffuse fluids with low concentrations. There are two larger groups of samples, distinguished by two different amino acid (AA) patterns: Group 1 has low concentrations in general, but relatively high concentrations of aspartic acid and glutamic acid, whereas group 2 has high abundances of aspartic acid, glutamic acid, serine, glycine, and alanine. The samples of the two groups do not share any other similarities, with the exception of their DFAA pattern. Thus, it appears that neither factor, such as fluid type or degree of dilution, influences the DFAA pattern, nor is the pattern site-specific. The presence of elevated AA concentrations in hydrothermal fluids implies that they are available as a nutrient source to vent organisms and as potential complexing agents for metals, possibly influencing their bioavailability and solubility.