detailed variations in bioactive elements in the surface ... · the manifold configurations, flow...

surface ocean, our research group hasaimed to develop and apply advanced tech-nologies to identify in detail the forms ofnutrients, organic matter and the microbio-logical community, and to detect their de-tailed variation. Specifically, the follow-ing four topics (sub-themes) were studied:(1) detailed analyses of the dynamics ofnutrients in the oligotrophic surface oceanusing a high-sensitive colorimetric method(J. Kanda and F. Hashihama); (2) the de-velopment of an accurate and simplemethod for measuring particulate phospho-rus and the investigation of the decompo-sition processes of dissolved organic phos-phorus in terms of hydrolysis extoenzyme(M. Suzumura); (3) the development andexamination of the method for measuringthe concentrations of volatile organic car-bon in seawater, and the characterizationof decomposition processes of dissolvedorganic matter (H. Ogawa); and (4) theelucidation of the microbial degradationmechanism of submicron particles in theocean (K. Kogure).

Topic 1: Detailed analyses of the dynam-

Detailed Variations in Bioactive Elements in the SurfaceOcean and Their Interaction with Microbiological Processes

H. Ogawa1*, K. Kogure1, J. Kanda2, F. Hashihama2 and M. Suzumura3

1Atmosphere and Ocean Research Institute, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8564, Japan2Department of Ocean Sciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan3National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba AIST West, 16-1 Onogawa, Tsukuba 305-8469, Japan*E-mail: [email protected]

Keywords: Marine Biogeochemical Cycles; Nutrients; Marine Organic Matter; Marine Microorganisms

General Introduction

For a deep understanding of the way ofbiogeochemical cycles between the surfaceocean and the lower atmosphere are linked,an important key is to elucidate the cyclesof bioactive macro elements, especially C,N, P, Si, through the biological processesin the surface ocean. The cycles of theseelements are driven by the inter-conver-sion between CO2 plus nutrients and or-ganic matter, however, detailed mecha-nisms still remain mostly unknown. Forexample, in the oligotrophic ocean, typi-cally in the subtropical regions, the mecha-nism of nutrient supplies which sustain theprimary production is not consistently un-derstood. The real forms of the bioactiveelements which are incorporated into or-ganic matter are not exactly identified, and,furthermore, their decomposition proc-esses still remains a “black box”. Conven-tional research approaches only are insuf-ficient to solve these subjects, and it isnecessary to introduce new technologies.Thus, in order to elucidate the mechanismof the cycles of bioactive elements in the

Western Pacific Air-Sea Interaction Study,Eds. M. Uematsu, Y. Yokouchi, Y. W. Watanabe, S. Takeda, and Y. Yamanaka, pp. 177–197.© by TERRAPUB 2014.

doi:10.5047/w-pass.a03.002

178 H. OGAWA et al.

ics of nutrients in the oligotrophic surfaceocean using a highly-sensitive colorimetricmethod (J. Kanda and F. Hashihama)

Introduction

In spite of the long history ofbiogeochemical studies at sea, our under-standing of nutrient dynamics in the sub-tropical oligotrophic ocean has been con-siderably limited. This limitation appar-ently derived from the low concentrationsof surface nutrients in the ocean, which arefrequently below the detection limits ofstandard analytical methods. In order toreveal surface nutrient dynamics in theoligotrophic ocean, we need to design newanalytical tools, and then to conduct newfield observations using these tools. Dur-ing this project, we developed a four-chan-nel highly-sensitive analytical system todetermine nanomolar concentrations ofnitrate+nitrite (N+N), nitrite, soluble re-active phosphorus (SRP), and silicic acid,and we examined the surface distributionof these nutrients, and associatedbiogeochemical processes, over theoligotrophic waters of the Pacific and In-dian Oceans.

Methods

Development of highly-sensitive colorim-etry

A four-channel highly-sensitive nutri-ent analyzer was constructed by connect-ing a Liquid Waveguide Capillary Cell(LWCC, World Precision Instruments) toa gas-segmented continuous flowcolorimetric system (AutoAnalyzer II,Technicon). The LWCCs have a long path-length of 50 cm for N+N and nitrite, and100 cm for SRP and silicic acid, respec-tively. The manifold configurations, flowdiagrams, and preparation of analyticalregents were based on the methods ofWhitledge et al. (1981) for N+N and ni-trite, and Hansen and Koroleff (1999) for

SRP and silicic acid. The detection limit,defined as three times the standard devia-tion of the blanks, was 2 nM for nitrite, 3nM for both N+N and SRP, and 11 nM forsilicic acid. Seawater collected from thesurface of the subtropical western NorthPacific, and preserved for >1 year, wasused as N+N and nitrite blanks, whoseN+N and nitrite concentrations were de-termined by a chemiluminescent technique(Garside 1982; Kanda et al. 2007). Theblanks for SRP and silicic acid were pre-pared by removing SRP and silicic acidfrom the western North Pacific surfacewater, based on the MAGIC (magnesium-induced co-precipitation) procedure (Karland Tien 1992). More detailed informationon the highly-sensitive colorimetry is de-scribed in Hashihama (2007) for N+N andSRP, and in Hashihama and Kanda (2010)for silicic acid.

Field observationWe conducted a continuous underway

survey on cruise transects of the R/VHakuho-maru, R/V Tansei-maru, and RT/V Umitaka-maru. These transects covereda broad area over the central and westernPacific Ocean and southern Indian Ocean.The survey was intermittently performedduring the period from December 2004 toSeptember 2008. This period includes aperiod prior to the project in order to inte-grate as much nutrient data as possible.During the project, a four-channel highly-sensitive nutrient analyzer was used tocontinuously measure the concentrationsof N+N, nitrite, SRP, and silicic acid inseawater, which was pumped up from theship’s bottom. Prior to this project, a two-channel highly-sensitive analyzer for N+Nand SRP was adopted. The spatial resolu-tion of our nutrient analysis was at a hori-zontal scale of approximately 1 km at acruising speed of 15 knots (27.8 km h–1).Temperature, salinity, and in vivo chloro-phyll fluorescence of the water were con-tinuously measured by using a

Bioactive Elements and Microbiological Processes 179

thermosalinometer, and a fluorometer,throughout all cruises. Data for nutrient,temperature, salinity, and in vivo chloro-phyll fluorescence were taken at 1-minintervals.

Along with the nutrient analysis,seawater samples were discretely collectedfrom the pumped-up water for total dis-solved phosphorus (TDP) analysis, micro-scopic observations, flow cytometry, andhigh-performance liquid chromatography(HPLC) algal pigment analysis. The TDPsamples were filtered through a pre-combusted Whatman GF/F filter by grav-ity filtration, and the filtrate was immedi-ately frozen for later analysis. The concen-tration of TDP was determined by apersulfate oxidation method using an au-tomated flow analytical system (QuAAtro,SEAL Analytical). The standard analyti-cal protocol of the manufacturer wasadopted. The concentration of dissolvedorganic phosphorus (DOP) was determinedby subtracting the SRP concentration fromthe TDP concentration.

The microscopic samples were fixedwith glutaraldehyde, or neutralized forma-lin, at a final concentration of 1% (v/v).The fixed samples were concentrated bysedimentation. Micro-sized diatoms anddiazotrophs (Trichodesmium and Richelia)in the concentrates were counted using aninverted microscope.

The samples for flow cytometry werefixed with 1% (v/v) glutaraldehyde. Thefixed samples were frozen in liquid nitro-gen and then stored at –80∞C until analy-sis. We used a PAS-III flow cytometer(Partec) equipped with a 488-nm argon ionlaser. Prochlorococcus, Synechococcus,and eukaryotes were identified usingprotocols established by Marie et al.(1999). In addition to identifying thesephytoplankton groups, we identifiednanoplanktonic unicellular cyanobacteriausing the procedure described by Kitajimaet al. (2009) and Sato et al. (2010). Thisnano-sized organism could potentially fix

dinitrogen gas under nitrate-depleted sur-face waters (Zehr et al. 2001).

The seawater samples for HPLC werefiltered through a Whatman GF/F filter.The filters were immediately frozen in liq-uid nitrogen and stored at –80∞C untilanalysis. Pigment analysis was conductedby the method described by Zapata et al.(2000) using a Shimadzu HPLC system.Ten biomarker pigments; monovinyl chlo-rophyll a , divinyl chlorophyll a ,pheophytin a, pheophorbide a, peridinin,19¢-butanoyloxyfucoxanthin, fucoxanthin,1 9 ¢ - h e x a n o y l o x y f u c o x a n t h i n ,myxoxanthophyll, and zeaxanthin, weredetected using a photodiode array. Thesepigments were quantified from the peakarea calibrated against that of standard pig-ments (DHI Water and Environment).

Results and Discussion

Basin-scale distribution of surface N+Nand SRP

The study area through the central andwestern Pacific Ocean, and the southernIndian Ocean, except for equatorialupwelling, was characterized by an over-all depletion of N+N concentration below20 nM (Fig. 1(a)), as has been reported inHashihama et al. (2009). Although allcruises were conducted during differentseasons, a consistent depletion of the N+Nconcentration was observed. Since nitriteconcentrations were nearly equal to thedetection limit of 2 nM, N+N concentra-tions were mostly ascribed to nitrate con-centration.

In contrast, SRP concentrations varieddynamically between <3 nM and 300 nMthroughout the study area (Fig. 1(b)). TheSRP concentrations were generally lower(<50 nM) in the western North Pacific andthe western Indian Ocean, and in the vi-cinity of Fiji, Hawaii, and Java, than inother areas including the western equato-rial Pacific, the Coral Sea, and the centralIndian Ocean (100–300 nM). Among the

180 H. OGAWA et al.

Fig. 1. Surface distribution of (a) N+N, and (b) SRP, in the central and western Pacific Oceanand the southern Indian Ocean.

lower SRP regions, a remarkable excep-tion was found: an almost complete ex-haustion of SRP (<10 nM) existed at a hori-zontal scale of >2000 km in the westernNorth Pacific in both summer and winter.The SRP exhaustion was most likely asso-ciated with an elevated dinitrogen fixationand nanocyanobacteria abundance(Kitajima et al. 2009). We suggest that themacro-scale SRP exhaustion with an en-hanced diazotrophic activity was driven byhigh dust deposition from the Asian conti-nent to the surface of the western NorthPacific (Hashihama et al. 2009). Since wealso observed a relatively high abundanceof Trichodesmium , Richelia , and

nanocyanobacteria in other lower SRP re-gions as described below, SRP decreasesin the surface waters of the subtropicalocean are suggested to be tightly coupledwith diazotrophic activity. On the otherhand, the higher SRP waters could be con-servative due to an inadequate dust sup-ply and little diazotrophic activity.

Diazotrophs can utilize several formsof DOP as an alternative to SRP (Dyhrmanet al. 2006), despite the fact that DOP isnot directly available for the most part.Surface DOP concentrations in the SRP-depleted western North Pacific were gen-erally above 200 nM and DOP was moreabundant than SRP. Although the signifi-


cance of the DOP pool as a source of P fordiazotrophs is still controversial (Moutinet al. 2008), we cannot exclude the possi-bility that the diazotrophic activity wassupported by the bioavailable DOP.

Sporadic decrease of SRP in the SouthPacific

In addition to the basin-scale variationof SRP, mesoscale SRP decreases at a<100-km horizontal scale were observedduring cruising. In particular, this was mostsignificant in the vicinity of the South Pa-cific islands during the R/V Hakuho-maruKH-04-5 cruise from January to February

2005 (Hashihama et al. 2010). In this area,a total of 17 transects, whose lengthsranged between 42 and 271 km, were sam-pled by continuous survey (Fig. 2(a)). Thisarea was characterized by an overall de-pletion of N+N as described above, butSRP varied from 7 to 192 nM.

In 7 out of the 17 transects, mesoscaleSRP decreases were observed. The SRPdecrease coincided with an elevation of invivo chlorophyll fluorescence, which wasaccompanied by an increase inphytoplankton abundance, as revealed bymicroscopy, flow cytometry, and accessorypigments. This mirror-image relationship

Fig. 2. (a) Cruise track in the vicinity of the South Pacific islands from January to February2005. Thick lines with numbers denote transects where a continuous underway survey wasdone. Surface distributions of temperature, salinity, N+N, SRP, chlorophyll fluorescence, andTrichodesmium on (b) Transect 5, and (c) Transect 11. Abundance of Trichodesmium is shownby black bars. Modified from Hashihama et al. (2010).

182 H. OGAWA et al.

between the SRP concentration andphytoplankton abundance was most appar-ent on both a 99-km transect east of Tonga,where the SRP concentration ranged from17 to 125 nM (Transect 5; Fig. 2(b)), andon a 98-km transect west of Fiji, where theSRP concentration ranged from 23 to 136nM (Transect 11; Fig. 2(c)). Both of thetransects contained distinct blooms ofTrichodesmium in areas with the lowestSRP concentrations. In contrast, no corre-sponding fluctuations in temperature andsalinity occurred in areas of minimum SRPlevels. The abundance of Trichodesmium,Prochlorococcus, Synechococcus, and allaccessory pigments examined tended to behigher in the low SRP waters than in thehigh SRP ones. In particular,Trichodesmium occurred in low SRP wa-ters (<25 nM). There was no significantrelationship between SRP concentrationsand nanocyanobacteria. These results sug-gest that surface SRP decreases are asso-

ciated with phytoplankton utilization ofSRP, and that the nitrogen supply fromTrichodesmium may contribute to this uti-lization.

Sporadic decrease of silicic acid in thewestern North Pacific

Continuous observations for trace sil-icic acid in conjunction with the N+N, ni-trite, and SRP observations, were per-formed in the surface waters of the west-ern North Pacific along the 138∞E lineduring the R/V Tansei-maru KT-07-15cruise from June to July 2007. As describedabove, the concentrations of N+N, nitrite,and SRP, were almost depleted in the west-ern North Pacific, while silicic acid con-centrations were generally above 500 nM.However, we detected an unstable field ofsilicic acid and associated diatom dynam-ics in the northern edge of the KuroshioCurrent during summer (Fig. 3(a)). Silicicacid concentrations between 33∞N and

Fig. 3. (a) Cruise track in the western subtropical North Pacific from June to July 2007. A lightgray arrow denotes the Kuroshio Current path, which was available from Quick Bulletin ofOcean Condition, published by the Hydrographic and Oceanographic Department of the Ja-pan Coast Guard (http://www1.kaiho.mlit.go.jp/KANKYO/KAIYO/qboc/index_E.html). (b) Sur-face distributions of N+N, SRP, silicic acid, and diatoms, on the 138∞E line.


34∞N along the 138∞E transect showedhighly-dynamic variations (Fig. 3(b)). Athreshold concentration for net silicic aciduptake by silicon-starved diatoms is gen-erally assumed to be approximately 1 mM(=1000 nM) (Paasche 1973). However, wedetected a complete depletion of <11 nMat a 4-km horizontal scale around 33.1∞N,and this concentration level was far belowthe threshold. Concentrations of N+N andSRP at the silicic acid depleted site werenot so much different from those in theneighboring waters. A sporadic N+N eleva-tion up to 145 nM was detected at 33.4∞N.The temperature and salinity did not ex-actly correspond to these nutrient fluctua-tions.

At the silicic acid depleted site, celldensities of diatoms drastically increasedup to 1358 cells l–1. This diatom bloommostly consisted of Chaetoceros spp. Incontrast to the cell abundance, a diatommarker, fucoxanthin, was quite low at thesilicic acid depleted site. This suggests thatthe synthesis of photosynthetic pigmentswas limited. Indeed, the concentration ra-tio of the chlorophyll derivative(pheophytin a + pheophorbide a) to chlo-rophyll a was apparently high at the sil-icic acid depleted site, implying that or-ganic matter in the cells was toward deg-radation rather than synthesis. These re-sults indicate that diatoms could exhaustsilicic acid up to an extremely-low con-centration level and that their cells werein a decline phase.

Conclusions

Our intensive observations first dem-onstrated the surface distribution ofnanomolar nutrients in the central andwestern Pacific Ocean and the southernIndian Ocean. The main findings are asfollows: (1) A basin-scale extremely-lowP availability appeared to be unique to thewestern North Pacific. (2) Mesoscale de-creases of the surface SRP, observed in thevicinity of islands, were likely producedthrough SRP uptake by phytoplankton, andsporadic Trichodesmium blooms mighthave played a significant role in the sup-ply of new nitrogen for SRP uptake. (3)Our highly-sensitive colorimetry coulddetect dynamic fluctuations of silicic aciddespite relatively consistent N+N, nitriteand SRP, and the unusual complete deple-tion of silicic acid was ascribed to a likelyuptake by diatoms. The highly-sensitivenutrient analysis is found to be essentialto biogeochemical studies in oligotrophicopen-ocean systems. Further studies usingthis technique should reveal various typesof temporal and spatial variations ofnanomolar nutrients and their interrelation-ships with biogeochemical processes.

Acknowledgements

We are grateful to Prof. K. Furuya for his total sup-port of this study. We thank the officers, crew mem-bers, and members of the scientific party of thecruises of R/V Hakuho-maru, R/V Tansei-maru, andRT/V Umitaka-maru, for their cooperation at sea.

References

Dyhrman ST, Chappell PD, Haley ST, Moffett JW, Orchard ED, Waterbury JB, Webb EA (2006)Phosphonate utilization by the globally important marine diazotroph Trichodesmium. Nature 439:68–71.

Garside C (1982) A chemiluminescent technique for the determination of nanomolar concentrationsof nitrate and nitrite in seawater. Mar. Chem. 11: 159–167.

Hansen HP, Koroleff F (1999) Determination of nutrients. p. 159–228. In Methods of Seawater Analysis(eds. Grasshoff K, Kremling K, Ehrhardt M), Wiley, Weinheim.

Hashihama F (2007) Study on nutrient and phytoplankton dynamics in the oligotrophic ocean. Ph.Dthesis, The Univ. of Tokyo, Tokyo, 138 pp. (in Japanese).

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Hashihama F, Kanda J (2010) Automated colorimetric determination of trace silicic acid in seawater bygas-segmented continuous flow analysis with a liquid waveguide capillary cell. La mer 47: 119–127.

Hashihama F, Furuya K, Kitajima S, Takeda S, Takemura T, Kanda J (2009) Macro-scale exhaustion ofsurface phosphate by dinitrogen fixation in the western North Pacific. Geophys. Res. Lett. 36:L03610, doi:10.1029/2008GL036866.

Hashihama F, Sato M, Takeda S, Kanda J, Furuya K (2010) Mesoscale decrease of surface phosphateand associated phytoplankton dynamics in the vicinity of the subtropical South Pacific islands.Deep-Sea Res. I 57: 338–350.

Kanda J, Itoh T, Nomura M (2007) Vertical profiles of trace nitrate in surface oceanic waters of theNorth Pacific Ocean and East China Sea. La mer 45: 69–80.

Karl DM, Tien G (1992) MAGIC: A sensitive and precise method for measuring dissolved phosphorusin aquatic environments. Limnol. Oceanogr. 37: 105–116.

Kitajima S, Furuya K, Hashihama F, Takeda S, Kanda J (2009) Latitudinal distribution of diazotrophs andtheir nitrogen fixation in the tropical and subtropical western North Pacific. Limnol. Oceanogr. 54:537–547.

Marie D, Partensky F, Vaulot D, Brussaard S (1999) Enumeration of phytoplankton, bacteria, and vi-ruses in marine samples. 11.11.1–11.11.15. In Current Protocols in Cytometry Supplement 10 (eds.Robinson JP, Darzynkiewicz Z, Dean PN, Orfao A, Rabinovitch PS, Stewart CC, Tanke HJ, WheelessLL, Dressier LG), Wiley, New York.

Moutin T, Karl DM, Duhamel S, Rimmelin P, Raimbault P, Mooy BASV, Claustre H (2008) Phosphateavailability and the ultimate control of new nitrogen input by nitrogen fixation in the tropical Pa-cific Ocean. Biogeosciences 5: 95–109.

Paasche (1973) Silicon and the ecology of marine plankton diatoms. II. Silicon-uptake kinetics in fivediatom species. Mar. Biol. 19: 262–269.

Sato M, Hashihama F, Kitajima S, Takeda S, Furuya K (2010) Distribution of nano-sized cyanobacteria inthe western and central Pacific Ocean. Aquat. Microb. Ecol. 59: 273–282.

Whitledge TB, Malloy SC, Patton CJ, Wirick CD (1981) Automated Nutrient Analysis in Seawater.Brookhaven Natl. Lab., New York.

Zapata M, Rodriguez F, Garrido JL (2000) Separation of chlorophylls and carotenoids from marinephytoplankton: a new HPLC method using a reversed phase C8 column and pyridine-containingmobile phases. Mar. Ecol. Prog. Ser. 195: 29–45.

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compounds in seawater is largely depend-ent on their chemical forms. While ortho-phosphate is known to be an overwhelm-ingly important P compound due to its highbioavailability, other forms of P includingparticulate, and dissolved, organic P (POPand DOP) have been considered as poten-tial P sources. However, few studies havebeen conducted on the spatial and tempo-ral variability of particulate P pools inpelagic environments (Loh and Bauer2000; Suzumura and Ingall 2004;Yoshimura et al. 2007). Particulate organiccarbon (POC) and nitrogen (PON) are fre-quently measured as a part of routinechemical analysis in marine observations,whereas POP determination is not often

Topic 2: Development of an accurate andsimple method for measuring particulatephosphorus and investigation of decompo-sition processes of dissolved organic phos-phorus in terms of hydrolysis extoenzyme(M. Suzumura and coworker: N. Yamada)

Introduction

Phosphorus (P) is the essential nutri-ent sustaining marine primary production.Regeneration of bioavailable dissolved Pfrom particulate matter is an importantprocess maintaining P availability in themarine ecosystem (Karl and Björkman2002; Paytan et al. 2003; Benitez-Nelsonet al. 2007), and the bioavailability of P


included. This is most likely because a POPdetermination requires an additional vol-ume of sample water as well as additionalanalytical procedures and costs. Also, asto the dissolved fraction, little is knownabout the chemical composition of DOP.Like C and N, P is the key element in themarine biogeochemical cycle. It is an im-portant issue to identify, quantify and char-acterize organic P in marine environmentsfor a better understanding of the dynam-ics of bioelements in the surface oceanecosystems.

The objective of this study was to makea breakthrough in the study of marine P.One approach was to develop a newmethod for the determination of POP. Theconventional high temperature dry com-bustion (HTDC) methods show high recov-eries (100 ± 5%) of standard organic Pcompounds (Solözano and Sharp 1980;Nedashkovskiy et al. 1995; Kérouel andAminot 1996; Ormaza-González andStatham 1996; Monaghan and Ruttenberg1999). In contrast, persulfate chemical wetoxidation (CWO) methods show low P re-coveries varying greatly among theprotocols (Kérouel and Aminot 1996;Monaghan and Ruttenberg 1999;Raimbault et al. 1999; Lampman et al.2001). The CWO method, however, is un-questionably simpler and less time con-suming than the HTDC method. Weighingthis advantage against the fact that CWOcan produce low and variable P recover-ies, a persulfate CWO method has beenimproved in this study and then comparedwith a conventional HTDC method.

Another approach was to examine theeffects of environmental conditions on theprocess of organic P regeneration in theocean surface ecosystems. In the regenera-tion of organic nutrients, enzymatic hy-drolysis is an essential process sustainingprimary productivity in the ocean. For ex-ample, alkaline phosphatase (AP) is themajor contributor to the remineralizationof organic P to liberate orthophosphate

(Hoppe 2003; Dyhrman and Ruttenberg2006 and cited therein). In this study, theeffects of pH variation on AP activitieswere examined by laboratory experiments,since pH is a particularly important factorregulating enzyme activities, and it is nowevident that increases in atmospheric CO2concentration have changed pH of oceansurface waters (Caldeira and Wickett2005). Also, field observations and ship-board experiments were executed to inves-tigate the depth profiles of various dis-solved P pools and AP kinetic parametersin the euphotic zone across the coastal tooceanic environmental gradient in thewestern North Pacific Ocean.

Methods

Particulate P determinationThe HTDC method used in this study

was based on the protocols of Aspila et al.(1976) and Solözano and Sharp (1980).Samples were added with Mg(NO3)2 andcombusted at 470∞C for 90 min. Phosphatewas extracted with 1 M HCl. In the CWOmethod tested here, samples were addedwith K2S2O8 solutions prepared from alow-P source (Wako Chemical, Japan) andranging from 0.5% to 3% (w/v) K2S2O8.The samples were then heated at 120∞C for30 min using an autoclave. Soluble reac-tive P concentrations in the HTDC extractand CWO solution were measured bymeans of a standard colorimetric method.The HTDC and CWO methods were com-pared using the commercially-available Pcompounds, and various natural analoguesof particulate matter including riverinesuspended particle matter (SPM), coastaland pelagic marine sediments, net-col-lected and cultured plankton, geochemicalreference materials, and estuarine andpelagic SPM.

Laboratory and field experiments for APkinetic studies

Acidification experiments for enzyme

186 H. OGAWA et al.

activities were carried out using surfaceseawater samples collected from four lo-cations from a highly-eutrophic site of theinnermost part of Tokyo Bay, the transi-tion area between coastal and open oceanenvironments in Sagami Bay and SurugaBay, and the oligotrophic open-ocean en-vironment outside the Kuroshio Current.Among them, AP activity was measured inthe samples from Tokyo Bay and SagamiBay. Enzyme activities were determined asthe hydrolysis rates of fluorogenicsubstrates (methylumbelliferyl phosphate(MUF-P)) according to the protocols ofprevious studies (Hoppe 1983; Fukuda etal. 2000).

Field observations and shipboard ex-periments to investigate various dissolvedP-pools and AP kinetic parameters wereconducted during the KT10-13 cruise ofthe R/V Tansei-maru in July 2010. Sam-ples were collected from a coastal stationin Sagami Bay and three pelagic stationsalong a transect outside the Kuroshio Cur-rent. Vertical profiles of SRP, DOP and APhydrolyzable DOP (APH-DOP) in theeuphotic zone were investigated using themethod of Suzumura et al. (1998). To ob-

tain complete kinetic parameters of thehydrolytic reaction via AP activity, multi-concentration measurements of MUF-Pfrom 50 to 1000 nM equivalent to the insitu DOP level were executed (Thingstadet al. 1993; Sebastián and Niell 2004).


Development of the CWO method forparticulate P determination

The recoveries of P relative to those ofthe HTDC method were 102 ± 6.7% fromstandard organic, and inorganic, P com-pounds, and 100 ± 3.8% from naturalparticulate matter analogues includingriverine suspended particulate matter(SPM), sediments, plankton, andgeochemical reference materials of rock.However, low recoveries of 14% to 69%were observed for reference samples ofclay minerals.

A comparison, using many samples ofestuarine and pelagic SPM, showed thatthe CWO method produced PP values con-sistent with those obtained by the HTDCmethod, except for some estuarine samplesenriched in inorganic matter (Figs. 4(a) and

Fig. 4. (a) Plots of particulate P concentrations of HTDC versus CWO in the estuarine sam-ples. The dashed line represents CWO/HTDC = 1. (b) Plots of particulate P concentrations ofHTDC versus CWO in the pelagic samples. The dashed line represents CWO/HTDC = 1. Re-printed from Suzumura (2008), Limnol. Oceanogr. Methods, 6, 626, copyright (2008) by theAssociation for the Sciences of Limnology and Oceanography, Inc.


(b)). The low recoveries were ascribed tothe presence of an inorganic P fractionhighly recalcitrant to chemical digestion.The procedural blank was lower for theCWO method than for the HTDC method.The filter blanks showed a large variationamong the fil ter materials tested;aluminum oxide membrane filters wereshown to contain a considerable amountof P that could cause significant contami-nation in both methods. Analytical preci-sion was equivalent between the two meth-ods. Considering its simplicity and its lesstime-consuming nature, the CWO methodpresented here is suitable for PP determi-nation in aquatic environments (Suzumura2008).

AP activity in various environmental con-ditions

AP activities showed relatively minorvariations over the pH range tested. How-ever, the trends of acidification effects onAP activity were opposite at the sites lo-cated in Tokyo Bay and Sagami Bay. Withconfidence levels of 99%, AP activity in-creased slightly with acidification in To-kyo Bay, whereas it decreased in SagamiBay. The effect of acidification on AP ac-tivities differed between the coastal andsemipelagic samples, and this was likelydue to a freshwater influence at thenearshore station. As well as the effects ofacidification on AP activity, those on theactivities of leucine aminopeptidase, a-and b-glucosidase, and lipase, were exam-ined with being important promoters of thedegradation of marine organic matter, in-cluding proteins, carbohydrates, organicphosphorus compounds, and lipids. Theeffects of acidification on enzyme activityappear to vary depending on enzyme typeand location, but we conclude that acidifi-cation will cause changes in the cyclingof organic matter in marine ecosystems, inparticular to proteinous and lipid sub-stances (Yamada and Suzumura 2010).

In the field observations of various dis-

solved P-pools, it was found that SRP con-centrations at/above the Chl a maximumlayer (CML) depth were quite low aroundthe detection limit (20 nM) at all stations,suggesting a P deficient condition. Belowthe CML, to 200-m depths, SRP concen-trations increased rapidly with depth to107–194 nM at the pelagic stations and to1100 nM at the coastal station. DOP wasthe dominant P pool at/above the CMLaccounting for 62 to 100% of the total dis-solved P. The lowest concentration of DOP(30 nM) was found in the coastal surfacewaters (0- and 10-m depths) and, belowthis zone, DOP rapidly increased to 260nM. DOP depth profiles at the pelagic sta-tions showed minimum concentrations(48–81 nM) at the subsurface (10–20 m),increasing with depth more gradually thanthat at the coastal station. The concentra-tions of APH-DOP were low, ranging from20 to 54 nM. APH-DOP composed a con-siderable fraction of the total DOP (50–81%) only above the CML at the coastalstation where the lowest DOP concentra-tion was observed. APH-DOP was rathera minor constituent, accounting for 14–48% of the total DOP in the pelagic sam-ples. A large part of the ambient DOP inocean waters was unreactive to AP, and,hence, could be low in bioavailability as aP source.

In some depth samples from all sta-tions, AP kinetic parameters, including thepotential maximum hydrolysis rate(Vmax), the apparent half-saturation con-stant (Km), and the turnover time of APH-DOP (TA), were characterized to evaluatethe importance of DOP remineralization inthe P cycle. The highest Vmax in thepelagic stations was observed at the sur-face and rapidly decreased to constantlylow values throughout the samplingdepths. At the coastal station, the highestVmax was observed at the subsurface (10m) and gradually decreased with depth.Comparing the data at the CML depth,there were considerable differences across

188 H. OGAWA et al.

References

Aspila KI, Agemian H, Chau ASY (1976) A semi automated method for the determination of inorganic,organic and total phosphate in sediments. Analyst 101: 187–197.

Benitez-Nelson CR, O’Neill Madden LP, Styles RM, Thunell RC, Astor Y (2007) Inorganic and organicsinking particulate phosphorus fluxes across the oxic/anoxic water column of Cariaco Basin, Ven-ezuela. Mar. Chem. 105: 90–100.

Caldeira K, Wickett ME (2005) Ocean model predictions of chemistry changes from carbon dioxideemissions to the atmosphere and ocean. J. Geophys. Res. 110: C09S04, doi:10.1029/2004JC002671.

Dyhrman ST, Ruttenberg KC (2006) Presence and regulation of alkaline phosphatase activity in eukaryoticphytoplankton from the coastal ocean: Implications for dissolved organic phosphorusremineralization. Limnol. Oceanogr. 51: 1381–1390.

Fukuda R, Sohrin Y, Saotome N, Fukuda H, Nagata T, Koike I (2000) East-west gradient in ectoenzymesactivities in the subarctic Pacific: Possible regulation by zinc. Limnol. Oceanogr. 45: 930–939.

Hoppe H-G (1983) Significance of exoenzymatic activities in the ecology of brackishwater: measure-ments by means of methylumbelliferyl-substrates. Mar. Ecol. Prog. Ser. 11: 299–308.

Hoppe H (2003) Phosphatase activity in the sea. Hydrobiologia 493: 187–200.Karl DM, Björkman KM (2002) Dynamics of DOP. p. 249–366. In Biogeochemistry of Marine Dissolved

Organic Matter (eds. Hansell DA, Carlson CA), Academic Press, San Diego, London.Kérouel R, Aminot A (1996) Model compounds for the determination of organic and total phosphorus

in natural waters. Anal. Chim. Acta 318: 385–390.Lampman GG, Caraco NF, Cole JJ (2001) A method for the measurement of particulate C and P on the

same filtered sample. Mar. Ecol. Prog. Ser. 217: 59–65.Loh AN, Bauer JE (2000) Distribution, partitioning and fluxes of dissolved and particulate organic C, N

and P in the eastern North Pacific and Southern Oceans. Deep-Sea Res. I 47: 2287–2316.Monaghan EJ, Ruttenberg KC (1999) Dissolved organic phosphorus in the coastal ocean: Reassess-

ment of available methods and seasonal phosphorus profiles from the Eel River Shelf. Limnol.Oceanogr. 44: 1702–1714.

Nedashkovskiy AP, Khokhlov DA, Salikova NN, Sapozhnikov VV (1995) A comparison of two methodsfor total phosphorus determination in seawater (persulfate digestion by Koroleff-Valderrama anddigestion with magnesium nitrate). Oceanology 34: 498–502.

Ormaza-González FI, Statham PJ (1996) A comparison of methods for the determination of dissolvedand particulate phosphorus in natural waters. Water Res. 30: 2739–2747.

Paytan A, Cade-Menum J, Mclaughlin K, Faul KL (2003) Selective phosphorus regeneration of sinkingmarine particles: evidence from 31P-NMR. Mar. Chem. 82: 55–70.

Raimbault P, Diaz F, Pouvesle W, Boudjellal B (1999) Simultaneous determination of particulate or-ganic carbon, nitrogen and phosphorus collected on filters, using a semiautomatic wet-oxidationmethod. Mar. Ecol. Prog. Ser. 180: 289–295.

Sebastián M, Niell FX (2004) Alkaline phosphatase activity in marine oligotrophic environments: impli-cations of single: substrate addition assays for potential activity estimations. Mar. Ecol. Prog. Ser.277: 285–90.

Solözano L, Sharp JH (1980) Determination of total dissolved and particulate phosphorus in naturalwaters. Limnol. Oceanogr. 25: 754–758.

Suzumura M (2008) Persulfate chemical wet oxidation method for the determination of particulatephosphorus in comparison with a high-temperature dry combustion method. Limnol. Oceanogr.

the environmental gradient from coastal toopen-ocean stations. The in situ hydroly-sis rate that was estimated from the ambi-ent APH-DOP concentration and TA de-creased considerably, whereas TA and Kmincreased. Although the concentrations ofDOP and APH-DOP were relatively com-parable across the environmental gradient,

a high efficiency in DOP remineralizationby AP activities observed only in thecoastal environment, could sustain P re-quirement from the high Chl a biomasstherein. The role of DOP as a potential Psource is quite complicated and its impor-tance varies greatly among the environ-mental conditions.


Methods 6: 619–629.Suzumura, M, Ingall ED (2004) Distribution and dynamics of various forms of phosphorus in seawater:

insights from field observation in the Pacific Ocean and a laboratory experiment. Deep-Sea Res. I,51: 1113–1130.

Suzumura, M, Ishikawa K, Ogawa H (1998) Characterization of dissolved organic phosphorus in coastalseawater using ultrafiltration and phosphohydrolytic enzymes. Limnol. Oceanogr. 43: 1553–1564.

Thingstad TF, Skjoldal EF, Bohne RA (1993) Phosphorus cycling and algal-bacterial competition inSandsfjord, western Norway. Mar. Ecol. Prog. Ser. 99: 239–259.

Yamada N, Suzumura M (2010) Effects of seawater acidification on hydrolytic enzyme activities. J.Oceanogr. 66: 233–241.

Yoshimura T, Nishioka J, Saito H, Takeda S, Tsuda A, Wells ML (2007) Distributions of particulate anddissolved organic and inorganic phosphorus in North Pacific surface waters. Mar. Chem. 103: 112–121.

Topic 3-1: Development and examinationof the method for measuring the concen-trations of volatile organic carbon inseawater (H. Ogawa)

Introduction

Dissolved organic carbon (DOC) in theocean is a huge carbon reservoir in theEarth’s surface system. Its standing stockamounts to 680 Gt which is approximatelyequivalent to the pool of CO2 in the atmos-phere (760 Gt) or land biomass (570 Gt).Little is known about the chemical com-position and structure of DOC. Recently,the molecular-weight distribution of DOChas been investigated, and these studiesdemonstrated that low-molecular-weight(LMW) fraction (<1 k Da) accounts for amajor part of DOC (60–80% of the bulkDOC; Ogawa and Tanoue 2003). Howeverthe chemical composition at the molecu-lar level is entirely unknown. On the otherhand, some specific compounds dissolvedin seawater having LMW and a low boil-ing point have been determined as vola-tile organic compounds, such as DMS,halocarbon, methane and other hydrocar-bons. Although the carbon amount of thesespecific compounds in total is less than 1%of DOC, it is possible that there might bemore kinds of volatile compounds inseawater that remain still unidentified,considering that most of DOC is LMW. Ifit is true, our understanding of carbon flux

to the atmosphere from the surface oceanshould be dramatically changed. Thus, itis necessary to determine the bulk concen-tration of volatile organic carbon (VOC)in seawater, but, in fact, there have beenfew studies of this. About 30 years ago,one study tried to determine VOC concen-trations using a purge and trap method(Mackinnon 1979). It was concluded thatVOC accounts for 1–6% of the total or-ganic carbon (TOC). It appears that thisresult did not have a great impact on thetrend of research because, at that time, theproblem of climate change, such as globalwarming and its relation to the oceaniccarbon cycle, received less attention thanat present. However, the result means that10–40 Gt of the VOC pool exists in theocean, which exceeds considerably thebiomass carbon in the ocean (ca. 3 Gt) andcannot be bypassed in terms of the oce-anic carbon cycle. Furthermore, as the ana-lytical precision of carbon (as CO2) hasgreatly improved compared with that of 30years ago, it is possible that we can esti-mate the pool size of VOC in the oceanmore precisely. Thus, we have sought todevelop and examine the method for meas-uring the bulk concentrations of VOC inseawater.

Methods

First, a purge and trap method to ex-tract VOC components from seawater was

190 H. OGAWA et al.

tested. The analytical system was allhomemade on the basis of the previousstudy (Mackinnon 1979). The carbon con-centrations were measured by a non-dis-persive infrared analyzer (NDIR) afterconverting VOC into CO2 through high-temperature combustion. Next, a distilla-tion method was examined. It is based onthe measurement of TOC concentrationsin distillate from seawater by using a ro-tary evaporator. A TOC analyzer(Shimadzu TOC-5000) was used for TOCmeasurements.


Examination of the analytical methodA variety of analytical conditions for

the purge and trap method, such as samplevolume, materials of adsorbent, purgingtime and temperature, were examined.However, the analytical blank which wasobtained from the measurement for ultrapure water (Milli-Q) was often compara-ble to the values of seawater samples. Andthe recoveries for some standard com-pounds of low boiling points that werespiked into seawater, were not satisfactory.Finally, it was concluded that the purge andtrap method was difficult to apply to themeasurement of the bulk VOC in seawater.Next, the blank, and recovery by the dis-tillation method, were tested for ultra-purewater and some standard compounds, re-spectively. Consequently, the results weremuch better than the purge and trapmethod. So, in this study, the distillationmethod was considered acceptable formeasuring the bulk VOC concentrations inseawater. However it is possible that a veryvolatile fraction, that is rapidly purgeable,might be missed when the sample is acidi-fied and bubbled to purge CO2 in the TOCmeasurements. In contrast, relatively lessvolatile (semi-volatile) compounds, due totheir hydrophilic properties, might be in-cluded in this measurement because partof them are possibly recovered in the dis-

tillate from seawater. Thus, further exami-nation is necessary, and the definition andchemical significance of the organic frac-tion obtained from this method should beclearer. At least it may be interesting con-sidering it as a possible source of organiccompounds in rain water derived fromsweater distillation.

The vertical distributions of VOC in theocean measured by the distillation method

The measurement of VOC concentra-tions by the distillation method was firstapplied to the samples collected in the sub-tropical areas on the KH-06-2 expeditionof R/V Hakuho-maru. The VOC concen-trations varied relatively largely from 5 to20 mM in the surface layer (<100 m),whereas those in the deep waters between200–3000 m were relatively constantaround 10–15 mM. This suggests that bothproduction and consumption might occurin the surface layers and a biologically re-fractory fraction of VOC might remain inthe deep layers.

Topic 3-2: Characterization of decompo-sition processes of dissolved organic mat-ter (by H. Ogawa)

Introduction

The concentrations of DOC in theocean are higher and more variable in thesurface waters, ranging normally from 50to 90 mM, than in the deep waters (around35–45 mM). Previous studies using a 14Cdating approach to oceanic DOC havedemonstrated that the fraction of DOC thatexists with a uniform concentrationthroughout the deep ocean has a long life(up to 6,000 yrs), while that accumulatingonly in the surface layers has a relativelyshort residence time. The former is called“refractory DOC; R-DOC” and the latteris “semi-labile DOC; S-DOC”. Some re-cent studies have reported that the concen-trations of S-DOC vary significantly with


region. In particular, those in the subtropi-cal region, where strong stratification de-velops within the surface water column,are much higher than those in the high-lati-tudes or the equatorial regions with anupwelling system. This is explained by thehydrographic stability of surface watermass mainly regulating the concentrationsof S-DOC (Ogawa and Tanoue 2003).However, little is known about the regionalvariation in their quality, especially themicrobiological degradability. Dissolvedorganic matter (DOM) includes nitrogen(DON) and phosphorus (DOP) as well ascarbon (DOC), which are essential ele-ments for biogeochemical cycles in the

ocean. The decomposition of the semi-la-bile DOM (S-DOM) leads to the supply ofnutrients into the euphotic layers, as wellas CO2 production. Therefore, it is possi-ble that the microbial degradability of S-DOM might be one of the key factorswhich control biological production in theoligotrophic environments of the subtropi-cal region. Thus, this study has sought toelucidate the decomposition process of S-DOM in the surface ocean.

Methods

The samples obtained through the KH-05-2 cruise of the R/V Hakuho-maru in the

Fig. 5. Distribution of bacterially-degradable DOC in surface waters along the meridiansection of 160∞W in the central North Pacific.

192 H. OGAWA et al.

central North Pacific were used for thedecomposition experiment of DOM. Thesurface waters were collected along themeridian transect of 160∞W at intervals of1 degree between 54∞N and 10∞S. The sam-ple, filtered through a Whatman GF/F fil-ter, was poured into a glass ampoule andthen sealed with a torch. The sample fordetermining the initial concentrations ofDOC and DON was frozen immediately.

That for the degradation experiment wasincubated in the dark at room temperature,and then frozen after 1 month. The meas-urement of DOC and DON concentrationswas conducted with a high-temperaturecombustion method (Ogawa et al. 1999).Nutrients produced by the degradation ofDON and DOP during the incubation weremeasured with an autoanalyzer (AACS III,BRAN+LUEBBE).

Fig. 7. The changes in phosphate concentrations during the degradation experiment of sur-face seawater collected along the meridian section of 160∞W.

Fig. 6. The changes in nitrate concentrations during the degradation experiment of surfaceseawater collected along the meridian section of 160∞W.



The north-south distribution of the sur-face DOC concentrations along the merid-ian section of 160∞W showed a clear trendindicating that they were significantly high(70–80 mM) in the subtropical region com-pared with those in the subarctic region(60–70 mM, Fig. 5). The DOC reductionby bacterial respiration during the 1-monthincubation were 5–15 mM (10–25% of theinitial DOC) in the subtropical gyre, whileless than 5 mM (<5% of the initial) in thesubarctic region. It demonstrates that themicrobial degradability of S-DOC couldvary largely with regions and most of S-DOC accumulated in the subtropical sur-face could not be easily utilized by het-erotrophic microbes.

The degradations of DON and DOPwere evaluated from the production of ni-

trate (Fig. 6) and phosphate (Fig. 7), re-spectively. Overall, the degradability ofboth DON and DOP is relatively higher inthe subarctic than in the subtropical area;however, the difference was not so largeas compared with the case of DOC. In thesubtropical gyre, approximately 20–100nM (1–3% of the initial DON) of nitrateand 0–50 nM (<15% of the initial DOP)of phosphate were produced by microbialoxidation of DOM during the 1-month in-cubation. It suggests that DON and DOPwould be potentially important sources ofnutrients which support primary produc-tion in the oligotrophic environment of thesubtropical gyre. Furthermore, it is possi-ble that S-DOM in the subtropical regionmight function efficiently as the carbonfixation through a selective recycling ofnitrogen and phosphorus relative to car-bon.

References

Mackinnon MD (1979) The Measurement of the volatile organic fraction of the TOC in seawater. Mar.Chem. 8: 143–162.

Ogawa H, Tanoue E (2003) Dissolved organic matter in oceanic waters. J. Oceanogr. 59: 129–147.Ogawa H, Fukuda R, Koike I (1999) Vertical distributions of dissolved organic carbon and nitrogen in

the Southern Ocean. Deep-Sea Res. I 46: 1809–1826.

Topic 4: Microbial degradation ofsubmicron particles in the ocean (by K.Kogure and coworkers: Y. Seo, T. Suga,H. Kondo, M. Nishimura)

Introduction

In the surface layer of the ocean, thereare approximately one million prokaryo-tic cells. Most of them are present in a free-living state. In this layer, there are also socalled submicron particles (SMPs) (Koikeet al. 1990), of which concentrations usu-ally exceed that of prokaryotic cells by oneto two orders of magnitudes. Therefore,prokaryotic cells may constantly collide

with those SMPs. The origin, chemicalcomposition and fate of SMPs are, how-ever, not yet clear (Yamasaki et al. 1998),primarily because technically it is not easyto collect them, sort them depending onparticular characteristics and analyze themchemically and quantitatively. Therefore,current knowledge has been obtainedmostly by a particle counter or electronmicroscopy. It is assumed that the charac-teristics of SMPs may vary, depending onthe environments, season, and various bio-logical components. For instance, viralparticles are one component of SMPs, andtheir numbers may fluctuate with those ofthe host cells.

194 H. OGAWA et al.

SMPs are considered as part of DOCand, generally of a biological origin. Asthey may be composed of relatively labileorganic materials, they potentially serve asorganic substrates for heterotrophic marinemicroorganisms. The understanding of theorigin, turnover, and degradation mecha-nisms is quite important for clarifying thematerial cycles in the ocean water column(Ogawa and Tanoue 2003). So first, howdo prokaryotic cells degrade SMPs? Ac-cording to the textbooks, they are first de-graded into smaller molecules or buildingblocks (Obayashi and Suzuki 2005) out-side of the cells. This may be accomplishedby extracellular enzymes or enzymes as-sociated with the surface of the cells. Themolecular weight should be less than sev-eral hundred prior to the uptake. As a sec-ond step, degraded components are trans-ported into the cells through appropriatetransport systems that are generally quitespecific and selective.

Although this basic concept of micro-bial biodegradation has been well estab-lished, it was developed by the experimen-tal examinations conducted in the labora-tory, which is characterized by high con-centrations of both cells and organic mat-ter. It is easily assumed that the concen-trations of extracellular enzymes may behigh, so that they effectively degrade high-molecular compounds into smaller mol-ecules. However, the application of thisconcept to the natural environment is dif-ficult primarily because the concentrationsof both cells and organic compounds areby far less than those in the test tubes. Inthat case, the cells may have two potentialproblems: first, cells have to recognize thecompositions of organic compounds in or-der to synthesize proper enzymes, other-wise, cells may waste their energy and cel-lular components; and second, even if thecells synthesize a proper enzyme, the ex-tracellular release may lead to the diffu-sion of degraded molecules. If the harvestis smaller than the investment to synthe-

size enzymes, cells may not be able tomaintain themselves.

We proposed a new concept for themicrobial degradation of SMPs in theocean, i.e., the cells first retain SMPsaround the cells, synthesize proper en-zymes, degrade while SMPs are still re-tained on the cell surface, and uptake withminimum diffusion. By retaining on thecell surface, the cells may be able to rec-ognize the chemical compositions ofSMPs. This step is crucial for minimizingthe cellular energy required for the biodeg-radation and subsequent uptake. The pur-poses of this investigation were, first, tocollect prokaryotic cells that may poten-tially have the ability of “particle captur-ing” (PC) and second, to clarify the eco-logical significance of those PC prokaryo-tic cells.

Methods

Two methods, an optical method usingatomic force microscopy (AFM) and amicrobiological method, were employed.For AFM, seawater samples were fixedwith formaldehyde (1%, final conc.), fil-trated through GS (Millipore, pore size 0.2mm) and observed by AFM (mode. SPM-9500 J2, Shimadzu). The AFM wasequipped with a microfabricated and ox-ide-sharpened Si3N4 cantilever (OMCL-AC160TS-C1, Olympus) with a pyramidaltip and a force of 42 Nm–1. Prokaryoticcells were differentiated from non-livingparticles on the basis of their size, shapeand cross-section (Nishino et al. 2004).Cells with SPMs were counted and therelative numbers of particle-possessingcells among the total number of bacterialcells was determined. At least 100 cellswere observed in each sample. The lattermethod was employed for collecting cellswith the PC ability and subsequent com-munity structure analyses. Cells with PCability were collected by using a magneticseparation method (Seo et al. 2007). In


brief, particles of an appropriate size andcomposition were added into 1 mL of sam-ple sweater, gently mixed for one hour andcollected with a magnet. The cells withparticles were treated for DNA extraction,PCR amplification for 16S rDNA andDGGE analyses. The fluctuation of bacte-rial cells with PC ability were observed ina mesocosm experiment conducted at theInternational Coastal Research Center,Ocean Research Institute, the Universityof Tokyo (ICRC-ORI) in Otsuchi, IwatePrefecture from May to June in 2008. Twowater tanks were filled with 200 l of 200mm filtered seawater from each samplingstation and amended with inorganic nutri-ents (NaNO3, final conc. 44.1 mM andNaH2PO4, final conc. 4.4 mM). The twotanks were placed in an open air pool, towhich surface seawater of the bay was in-troduced to maintain the temperature. OnDay 5, both tanks were covered with ablackout curtain to accelerate the degra-dation process of phytoplankton. Theseawater samples were taken with acid-washed polycarbonate bottles after mixingthe seawater in the mesocosm.

Fig. 8. Community structure analyses of cells collected by using different paramagneticbeads. Each lane indicates a sample obtained with paramagnetic beads of different size andmaterials. The numbers indicate the size of particles. D; dextran, S; silica, T; total population, F;filtered samples.


Collection and characterization ofprokaryotic cells with PC ability

The cells with PC ability were collectedfrom seawater samples taken from TokyoBay, Sagami Bay, and offshore environ-ments by a magnetic beads method of dif-ferent sizes and materials. The relativenumbers of such cells, and their commu-nity structures were analyzed in compari-son with those of total cells. The resultsshowed the followings: first, generally thenumbers of prokaryotic cells collected withthe paramagnetic beads were approxi-mately one tenth of the total bacterial cells.Those cells are regarded as those whichhave the ability to absorb the paramagneticbeads around the cell surface during theone-hour incubation time. Therefore, it isregarded that those cells potentially havethe ability to capture SMPs. Second, thecommunity structures of those with PCability were analyzed by the DGGEmethod (Fig. 8). They were different fromthose of total cells in the seawater. Thisindicates that there are unique populationsthat have PC abilities in seawater. Third,

196 H. OGAWA et al.

the apparent community structures variedwith the paramagnetic beads used. Al-though both size and materials affected theresults, it seems the size was more influ-ential. For instance, when particles largerthan 6 mm were used, the apparent com-munity structures were considerably dif-ferent from those collected with particlesless than 250 nm. For prokaryotic cells, the6 mm particles may serve as a substrate forattachment, not capturing because they aremuch larger than the cell size. Therefore,this result indicates that abilities of attach-ment and capturing are not always same,and different populations might be in-volved in attachment or capturing. Fourth,the cell-specific aminopeptidase activitywas measured for the cells with PC abil-ity, and others. The activities of the formerwas generally 4 to 7 times higher thanthose of the latter, indicating that particle-capturing activity is coupling with high-degradation activities.

Observation of cells with PC ability byusing AFM

Although the method using magneticbeads provide us with information on

prokaryotic cells with possible PC ability,it is critical to observe cells directly andto confirm the occurrence of SMPs aroundthe cells. However, both electronmicroscopy and epifluorescent microscopyhave a limitation for this purpose. Tediouspreparations are required for the formerand such treatments may cause artifact.Although the sample preparation is mucheasier for the latter, the resolution is notsufficient for recognizing SMPs on thecell. Therefore, AFM was used for the ob-servation of cells and SMPs on them. Thefollowing results were obtained by thisapproach. First, the presence of prokaryo-tic cells possessing particles around thesurface in marine environments was con-firmed. Second, the relative numbers ofthose cells varied horizontally and verti-cally (Fig. 9). Generally relatively highnumbers were observed in highly eutrophicareas. In the open ocean, however, thenumbers were higher in deep layers (500–2,500 m). These results indicate that therelative numbers of prokaryotic cells withPC ability may reflect the concentrationand availability of organic matter in theenvironments. However, further investiga-tion is required to confirm this result.

Fig. 10. Change in the relative numberof prokaryotic cells with PC ability duringthe course of the mesocosm experiment.Three lines indicate results obtained withparticles of different size.Fig. 9. Relative number of cells with PC

abilities. T; Tokyo Bay, P; Sagami Bay, S;offshore stations.


Dynamic variation of prokaryotic cellswith PC ability in the mesocosm

The change in the number and commu-nity structure of cells with PC ability wasinvestigated by using meso-scale seawatertanks in Otsuchi Bay. The two tanks filledwith natural seawater were placed in anopen space and amended with nutrients toinduce phytoplankton bloom. The tankswere exposed to natural light at the begin-ning, and then covered with black curtainfor initiating degradation steps. The fieldobservations clarified the followings: First,the relative numbers of cells with PC abil-ity sharply increased following algalbloom, and then declined (Fig. 10). Sec-ond, the community structures of cells withPC ability were different from those oftotal populations in the mesocosm.

Conclusions

1. It was confirmed that by using para-magnetic beads and subsequent collectionwith a magnet, cells with the ability to cap-ture submicron particles are recovered

from natural seawater. Their communitystructures are different from those of thetotal populations, indicating that there arespecific groups with higher PC ability.

2. The relative numbers of cells withPC abilities varied horizontally and verti-cally in marine environments. In addition,the numbers also changed withphytoplankton bloom in the mesocosmtank. It is assumed that the numbers changewith the concentration of organic matter,quantity, and quality of SMPs and otherenvironmental parameters.

3. This is the first conceptual modelof how prokaryotic cells may degradeSMPs in marine environments. Further in-vestigation should clarify the mechanismof capturing and also the ecological sig-nificance of cells with PC abilities.

Acknowledgements

We are grateful to the Directors, researchers, cap-tains, and the crews, of the R/V Tansei Maru andalso the staff at ICRC-ORI (currently, ICRC-AORI).

References

Koike I, Hara S, Terauchi K, Kogure K (1990): Role of submicrometre particles in the ocean. Nature345: 242–243.

Nishino T, Ikemoto E, Kogure K (2004): Application of atomic force microscopy to observation ofmarine bacteria. J. Oceanogr. 60: 219–225.

Obayashi Y, Suzuki S (2005) Proteolytic enzymes in coastal surface seawater: Significant activity ofendopeptidases and exopeptidases. Limnol. Oceanogr. 50: 722–726.

Ogawa H, Tanoue E (2003): Dissolved organic matter in oceanic waters. J. Oceanogr. 59: 129–147.Seo Y, Ikemoto E, Yoshida A, Kogure K (2007): Particle capture by marine bacteria. Aquat. Microb.

Ecol. 49: 243–257.Yamasaki A, Fukuda H, Fukuda R, Miyajima T, Nagata T, Ogawa H, Koike I (1998): Submicrometer

particles in northwest pacific coastal environments: Abundance, size distribution, and biologicalorigins. Limnol. Oceanogr. 43: 536–542.

detailed variations in bioactive elements in the surface ... · the manifold configurations, flow...

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