Vol. 117:207-218, 1995MARINE ECOLOGY PROGRESS SERIES

Mar. Ec;pl. Prog. Ser.Published February 9

Community metabolism and nutrient cycling in theMississippi River plume: evidence for intense

nitrification at intermediate salinities

J. D. Pakulski 1, R. Benner1, R. Amon1, B. Eadie2, T. Whitledge1

I University of Texas at Austin Marine Science Institute, Port Aransas, Texas 78373, USA2Cooperative Institute for Umnology and Ecosystem Research, NOAA Great Lakes Environmental Research Laboratory,

2205 Commonwealth Blvd, Ann Arbor, Michigan 48105, USA

ABSTRACT: Community respiration, net nutrient fluxes and heterotrophic bacterial production wereinvestigated in the Mississippi River (USA) plume during May 1992 using dark bottle incubations ofunfiltered water. Highest rates of community O2 consumption and dissolved inorganic carbon regener-ation were observed at intermediate (10 to 27%0)plume salinities. Plume surface O2 consumption rateswere 2- to 4-fold greater than rates reported previously during the summer and winter. Heterotrophic

~__bacteriaLpmdur-tinn ([JH]_lpl1r-inpinr-nrpnr"tinn)w"c "tcoltighest-aUntermedilinities-aad-2-tG- __ _4-foldgreater than rates reportedfromother seasons.Net regenerationof NH/ was observedin theo to 18%0region of the plume while low rates of net NH4+ consumption were observed at 27 %0.Net N02-regeneration in the Mississippi River suggested the occurrence of nitrification in the fresh waters of thedelta. Serendipitous observations of rapid NOJ - regeneration at 18 and 27%0 indicated the develop-ment of intense nitrification at intermediate plume salinities. Nitrification accounted for 20 to >50% ofthe community O2 demand at 18 and 27%0. These data indicated that nitrification was an importantcomponent of the plume nitrogen cycle and contributed significantly to oxygen consumption in theplume.

- ---

KEYWORDS: Mississippi River plume. Bacteria. Nitrification' Respiration' Nutrient cycling

INTRODUCTION

Nutrient-rich water originating from the Mississippiand Atchafalaya Rivers (USA) supports high levels ofprimary production in the northern Gulf of Mexico(Riley 1937, Lohrenz et al. 1990). Historical data indi-cate that nutrient concentrations and ratios in the

Mississippi River have changed dramatically over thelast 40 years primarily as the result of increased fertil-izer use in the Mississippi River watershed (Turner &Rabalais 1991, Bratkovich & Dinnel 1992). It has beenhypothesized that anthropogenic increases in nutrientinputs from the Mississippi and Atchafalaya Rivershave enhanced primary production in adjacent coastalwaters. Sedimentation of organic matter derived fromnutrient-enhanced primary production, and strength-ened stratification of the water column in the summer

months, contribute to the seasonal formation of large

o Inter-Research 1995Resale of full article not permitted

areas of hypoxic «60 pM O~) bottom water on theinner Louisiana shelf (Turner & Rabalais 1991). Theformation of these hypoxic waters adversely affects theabundance and diversity of fishes and benthic organ-isms in the areas affected by these events (Harper etal. 1981, Pavela et al. 1983, Gaston 1985, Renard 1986).

The discharge of the Mississippi River from the Mis-sissippi Delta results in the formation of a broad uncon-fined plume of low salinity water extending southwestfrom the delta along the inner Gulf shelf. MississippiRiver waters entering the Gulf of Mexico are charac-terized by high concentrations (50 to 100 pM) of nitrateand suspended particulate matter (-60 mg' I-I; Lohrenzet al. 1990). Due to the turbidity of the MississippiRiver, the distribution of primary production along theplume salinity gradient is influenced primarily by lightavailability at lower plume salinities and nutrient avail-ability at. higher salinities (Lohrenz et al. 1990). As a

208 Mar. EcoJ. Prog. Ser. 117: 207-218, 1995

result of the interaction of light and nutrient avail-ability along the plume salinity gradient; highestchlorophyll concentrations and rates of photosynthesiswithin the plume are observed at intermediate (10 to30'1'00)plume salinities (Lohrenz et al. 1990, Dagg &Whitledge 1991. Dortch et al. 1992a, Hitchcock & Whit-ledge 1992). As a consequence of the distribution ofprimary production across the plume salinity gradient,bacterial abundances and production (Chin-Leo &Benner 1992, Cotner & Gardner 1993) and mesozoo-plankton abundances (Dagg & Whitledge 1991) arealso highest at intermediate plume salinities, particu-larly in spring and summer. Study site and sampling procedures. Sample collec-

It has been hypothesized that N regeneration within tions and experiments were conducted from May 4the plume may greatly amplify the effect of N loading to 13, 1992, aboard the RV 'Longhorn'. Water samplesfrom the Mississippi River on the inner Louisiana shelf were collected along a transect originating at the(Turner & Rabalais 1991, Dortch et al. 1992a). The Head of Passes in the Mississippi River delta, throughoften nonconservative distribution of N and other Southwest Pass (a major distributary of the delta)nutrients across the plume salinity gradient suggests and extending southwest into the northern Gulf ofrapid uptake and cycling of these materials at inter- Mexico (Table 1). Plume samples were obtained withmediate salinities (Fox et al. 1987, Lohrenz et al. 1990, clean plastic buckets. Bucket samples were collectedDagg & Whitledge 1991). Rapid cycling of nutrients into a clean (2 N HC1, distilled H20 and sample rinsed)within the plume may influence nutrient ratios and the polyethylene carboy and mixed prior to experimentalspatial distribution of 'new' (N03--based) and 'regen- incubations. A visible surface diatom bloom waserated' (NH4+ based; D\l~g 1967) pri0 Pfesent at 27°~ NutrienLC:ilmpl/;'.. in thp npeD-.G.ulfmary production across the plume salinity gradient. of Mexico (36%0) were collected at 5 m using aVariation in nutrient ratios resulting from differential Niskin bottle equipped with teflon-coated springs.nutrient uptake and regeneration along the plume Sample salinities were measured with a Reichertsalinity gradient may further influence the size dis- refractometer.tribution or species composition of the phytoplankton Sample incubation. Mixed water samples were dis-community (e.g. fast-sinking diatoms vs slow-sinking pensed into clean (1 N HC1, distilled H20 and samplephytoflagellates tmd cyanobacteria) and consequently rinsed) 300 ml BOD (biological oxygen demand) bot-the flux of particulate organic matter from the plume lIes and incubated in the dark at ambient temperatureto the benthos (Dortch et al. 1992b). in a precision incubator (Fisher Model 146A). At 2 to

Bacteria contribute to both community respiration 6 h intervals during each experiment, bottles were(Chin-Leo & Benner 1992) and NH4+ regeneration removed from the incubator for nutrient, DIC and O2(Cotner & Gardner 1993) within the plume. Bacterial analyses. At each time point, 3 bottles were poisonedNH4+ regeneration rates are highest at intermediate with 50 111of saturated HgCl solution for DIC analysesplume salinities in summer (Cotner & Gardner 1993). and 3 to 5 bottles fixed for Winkler O2 determinations.Community NH4+ regeneration rates, however, can Nutrient concentrations were also determined from 3greatly exceed the rates of either N03 - or NH4+ up- bottles at each time point. Nutrient samples were fil-take, particularly at intermediate salinities (Dortch et tered through com busted glass 'fiber filters (Whatmanal. 1992a). GF/F) at low vacuum prior to analyses.

Organisms in the> 1.0 llm size fraction account for '

44 to 68 % of community O2 consumption at inter-mediate plume salinities in summer (Benner et al.1992), suggesting that bacteria may not be the princi-pal consumers of O2 within the plume. The high con-centrations of phyto- and mesozooplankton (Lohrenzet al. 1990, Dagg & Whitledge 1991) typically presentat intermediate salinities may thus contribute substan-tially to plume community O2 consumption. In addi-tion, there is some evidence for nitrification in the fresh

waters of the Mississippi River delta (Fox et al. 1987).The intensity of nitrification and its contribution to both

community O2 consumption and N cycling within theMississippi River plume, however, are not known. Inthis report, we present the results of short-term (12 to24 h) dark bottle incubation experiments in which O2consumption, dissolved inorganic carbon (DIC) pro-duction, bacterial abundance and production, andassociated net fluxes of Nand P were measured across

the salinity gradient of the Mississippi River plume.

MATERIALS AND METHODS

Table 1. Location, salinity, and temperature of MississippiRiver plume stations sampled during May 1992

Latitude Longitude Temperature(0C)

Salinity(%0)

29° 10.26' N28° 52.08' N28° 51.33' N28° 46.58' N27° 35.10' N

89° 15.72' W89° 28.31' W89° 31.61' W89° 30.42' W89° 57.49' W

18.920.921.021.922.4

o10182736

Pakulski et al.: Nutrient cycling in the Mississippi River plume 209

Oxygen consumption. Dissolved O2 concentrationswere measured by the Winkler method (Carpenter1965). A single 50 ml aliquot of fixed sample wasdrawn from each BOD bottle with a volumetric pipette(Oudot et al. 1988) and titrated with a 0.0125 N solutionof NaS20J' Titration equivalence points were deter-

'mined potentiometrically with a Mettler DL-21 auto-titrator equipped with a platinum combination elec-trode (Mettler DM 140-SC, Oudot et al. 1988, Graneli& Graneli 1991, Pomeroy et al. 1994). Standardswere prepared with commercially available 0.025 NKH(IOJh solutions (Fischer Scientific). Blanks wereequivalent to <1.5 pM dissolved O2 or <0.8% total dis-solved O2 concentrations. The precision (coefficient ofvariation) of the sample titrations was 2.4 % (n = 21time points). Oxygen consumption rates were deter-mined from the slope of the least-squares linearregression equation calculated for each time courseexperiment. All data points were included in eachregression. Analysis of variance for each regression(including DlC and nutrient data, see below) wasperformed to determine significant (p < 0.05) slopes.

Dissolved inorganic carbon determination. DIC wasmeasured by coulometry, using sample handling pro-cedures described by Dickson & Goyet (1991). Sampleswere transferred from BOD bottles to a strippingchamber pre filled with 4 ml prestripped 15 % HJP04.The sample was stripped of DlC into a coulometer cell(VIC, Inc., Model 5120) and automatically titrated to aconstant endpoint. Standards were prepared fromNa2COJ' Precision from replicate analysis of standardswas :t:2.6 pmol kg-I. Differences among triplicate watersamples were sometimes larger, presumably due to theheterogeneity in the water samples. DIC productionrates were determined from the slope of the least-squares regression line obtained from all time points ineach time course experiment.

Nutrient analyses and flux rates. Analyses for NH4+,

N02- and NOJ- were performed aboard ship with anAlpkem rapid flow analyzer according to the proce-dures of Whitledge et al. (1981). Soluble reactive phos-phorus (SRP) analyses were performed on thawedsamples in the laboratory. Net nutrient flux rates werecalculated from the slope of the least-squares regres-sion line from each time course experiment.

Lipshultz et al. (1986) reported that 15Nfluxes meas-ured in the Delaware River were often substantiaily

different between light and dark treatments. We didnot evaluate the effect of light on our measurements ofcommunity nutrient fluxes. We recognize that light-mediated reactions (e.g. photosynthesis) are importantmechanisms influencing the flux and concentrationsof dissolved materials in the plume. Dark reactions,however, are equally important on diel time scales.Moreover, in the turbid waters of the Mississippi River

j

jI...

plume, particularly at lower plume salinities wherelight limits photosynthesis, dark reactions may be thedominant factors influencing nutrient cycling. .

We further recognize that our estimates of net nutrientflux rates may underestimate absolute (gross) values. Inaddition, it should be noted that a determination of zeronet flux does not necessarily imply zero gross flux as up-take may balance production or regeneration.

Bacterial abundances and production. Bacterialabundances were measured by epifluorescence micro-scopy of DAPI-stained samples (Porter & Feig 1980).Bacterial production was determined by [JH]-leucineincorporation (Kirchman et al. 1985). Leucine incorpo-ration rates were determined at the beginning, middleand end of each time course experiment. At each timepoint, 10 ml samples from duplicate or triplicate BODbottles were amended with [JH)-leucine (New EnglandNuclear, Boston, MA, USA; 60 mCi mmol-I; 10 nMfinal concentration) and incubated for 30 min. Initial(t =0) incorporation rates were used to estimate in situbacterial production at the time of sampling and forcomparisons to other rate measurements. The meancoefficient of variation for triplicate bacterial produc-tion estimates was 24 % (n = 12). Bacterial production

estimates were obtained from highly turbid unfilteredsamples and sample heterogeneity contributed tohigher variances at some time points. Controls werekilled with formalin. Leucine incorporation rates werelinear up to 60 min of incubation but were not satu-rated with addition of 10 nM leucine.

Leucine incorporation rates were converted to bac-terial carbon assuming a conversion factor of 3.1 kgbacterial C produced per mole incorporated leucine(Simon & Azam 1989). Estimates of bacterial produc-tion using the latter conversion factor are comparableto those derived from thymidine incorporation and anempirically derived conversion factor for MississippiRiver plume bacteria (Chin-Leo & Benner 1992).Bacterial respiration rates were estimated assumingan empirically derived leucine incorporation-basedgrowth efficiency of 24 % for Mississippi River plumebacteria (Chin-Leo & Benner 1992).

RESULTS

Oxygen consumption and DIC production

Oxygen consumption and net DlC production rateswere not significant (p > 0.05) in the Mississippi River(Table 2). Leucine incorporation rates and net nutrientfluxes (below), however, indicated low but measnrablemicrobial activity at this station. Oxygen consumptionand DlC production rates measured in the 10 to 270/00region of the plume varied from 0.59 to 3.65 pM O2 h-1

210 Mar. Eco!' Prog. Ser. 117: 207-218, 1995

Table 2. Dissolved O2 consumption and dissolved inorganiccarbon (DIC) production rates from the Mississippi Riverplume during May 1992. Positive values indicate a netincrease and negative values a net decrease in O2 and DIC

concentrations

n' Not significant (p > 0.05)

and 0.87 to 2.91 pM C h-t, respectively (Table 2). BothDIC production and O2 consumption were greatest at18'1'00.

Plume heterotrophic bacterial production.respiration and abundances

Bacterial abundances across the plume salinity gradi-ent ranged from 3.1 to 9.1 x 105cells ml-1 (Table 3). Bac-terial cell densities were greatest at 10'1'00and declinedat higher salinities. Initial (t =0) bacterial production

rates ranged from 0.035 to 0.313 pM C h-1 (Table 2).Bacterial production rates generally increased duringthe course of the incubations (Fig. 1). The spatial distri-bution of bacterial production corresponded to thedistribution of O2 consumption and DIC productionacross the plume salinity gradient, with the highestbacterial production rates measured at 18'1'00(Table 3).Estimates of heterotrophic bacterial respiration rangedfrom 0.15 to 1.30 pM C h-1 (Table 3) and were equiva-lent to 18 to 45 % of net commuhity DIC production inthe 10 to 27 '1'00region of the plume (Table 3).

0.8 ---A- o%.

--0- 10%.18%.

-D- 27%.c~ 0.6u::I~'01o~

a. () 0.4iii~'55 E-'0II!

CD 0.2

0.0o 3 6

Time (h)

9 12

Fig. 1. Time course of bacterial production in experimentalincubations of unfiltered Mississippi River plume water,

May 1992

Plume nutrient concentrations and net nitrogenfluxes

Plume NH4+ concentrations varied from 0.29 to2.39 pM (Table 4) and exhibited enhanced concentra-tions at 10 and 27'1'00(Fig. 2). Net NHA+Egener~tionwas observed at lower plume salinities with thehighest rate recorded at 18'1'00(Table 5). In the 18%.experiment, NH4+ concentrations did not changeappreciably after 2 h of incubation. Net NH4+ regener-ation at 18'1'00(0.43 J.lM h-1) was estimated from thestatistically significant (p = 0.04, paired Hest) increasein NH4+ concentration between the time zero and the2 h time points. Net uptake of NH4+, however, wasobserved at 27'1'00(Table 5). The transition between thezones of net NH4+ regeneration and net NH4+ uptakewas coincident with the region of highest plume respi-ratory activity.

Nitrate exhibited non conservative behavior across

the plume salinity gradient with depressed concentra-tions at intermediate salinities (Fig. 2). Nitrate concen-trations were highest in the Mississippi River (114.6pM) and declined rapidly with increasing salinity.(Table 4). Net N03- uptake was observed in the Missis-

Table 3. Salinity, bacterial abundance, bacterial production, bacterial respiration, and bacterial respiration as percentage of netDIC production for stations sampled in the Mississippi River plume during May 1992. Bacterial respiration was estimated from

bacterial production rates assuming a growth efficiency of 24 % (Chin-Leo & Benner 1992)

Salinity Flux rate r2 p n Incubation time(%.) (pM h-1) (h)

O2 consumption0 +0.12n' 0.03 0.62 12 12

10 -0.59 0.77 <0.01 15 1218 - 3.65 0.17 0.05 21 1227 - 1.74 0.44 <0.01 19 12

DIC production0 _ 0.18n' 0.04 0.54 11 12

10 + 1.47 0.48 0.04 9 1218 + 3.00 0.87 < 0.01 9 1227 + 0.85 0.68 <0.01 9 24

Salinity Bacterial abundance Bacterial production Bacterial respiration Bacterial respiration as(%0) (105 cells ml-1) (pM C h-I) (JIM C h-1) % DIC production

0 4.98 0.071 0.2910. 9.12 0.158 0.66 4518 8.99 0.313 1.30 4327 7.54 0.035 0.15 18

Pakulski et al.: Nutrient cycling in the Mississippi River plume 211

9 18 27

Salinity (ppt)

120

100

36 9 18 27

Salinity (ppt)

36

b 1.5d

Fig. 2. Property salinity plots for (a)NH4+, (b) N02 -, (c) N03 -, and (d)soluble reactive P (SRP) from theMississippi River plume, May 1992.Dashed line connecting salinity endmembers approximates the con-

servative mixing distribution

~80~'.., 60oZ 40

20

oo 9 18 27

Salinity (ppt)

sippi River but there was not signlficant fluXOfN03 - at10%0 (Table 5). We observed high rates (0.37 and0.56 pM h-l) of net N03- regeneration at 18 and 27%0(Table 5).

Nitrite concentrations varied from 0.01 to 1.56 pM(Table 4) and exhibited enhanced concentrations atintermediate salinities (Fig. 2). A significant 'net regen-eration of N02- was observed in the Mississippi River(Table 5). No significant net fluxes of N02 -. however,were observed in the plume (Table 5).

SRP concentrations

SRP concentrations varied from 0.28 to 1.40 llM andwere highest in the Mississippi River (Table 6).Depressed SRP concentrations at intermediate salini-ties suggested a pronounced P-sink in the 18 to 27%0

~ 1.0~Cl.II:(/) 0.5

-". - ~"....................

...................

360.0

o 6 12 18 24 30 36

Salinity (ppt)

--regiOn of the plume (FIg. ~Net changes In SRPcon-centrations were insignificant (p > 0.05, paired t-test)over the course of all experimental incubations(Table 6).

---

Nutrient ratios

Molar dissolved inorganic N (DIN =NH4+ + N02 - +N03 -) to SRP ratios were high in the MississippiRiver (103:1) and increased to 183:1 at 18%0 beforedeclining to 64:1 at 27%0 (Table 4). The molarDIN: SRP ratio (5: 1) at 360/00,however, was substan-tially lower than those encountered in the plume.Molar N03 -: NH/ ratios varied considerably acrossthe plume salinity gradient but were higher (~26: 1)at salinities :5:18%0and substantially lower (:5:12:1) at27 and 360/00(Table 4).

3., a2,0l

c-

:r-\A1.5

+ . '" 1.0:I: 0Z Z

0.5

Table 4. Inorganic nutrient concentrations (%1 SD), and molar DIN(NH4+ + N02- + N03-):SRPand N03-: NH4+ ratios from theMississippi River plume during May 1992

Salinity NH4+ N02- N03- DIN SRP DIN: SRP N03-:NH4+(%0) (pM) (pM) (pM) (pM) (pM)

0 2.06 %0.31 0.37 %0.02 114.3 %0.12 116.7 1.13 %0.47 103 5510 2.39 %0.44 1.56 %0.43 62.0 %0.64 66.0 0.60 %0.15 110 2618 0.81 %0.38 1.01 %0.38 49.5 %0.56 51.3 0.28 %0.03 183 6127 1.73 %0.21 0.57 %0.21 16.2 %0.32 18.5 0.29 %0.05 64 936 0.29 %0.10 0.01 %0.00 3.4 %0.00 3.7 0.69 %0.05 5 12

212 Mar. Ecol. Prog. Ser. 117: 207-218, 1995

DISCUSSION

Nitrogen fluxes and nitrification in theMississippi River plume

As nitrification in the Mississippi River plume hadnot been reported previously, we did not anticipatethat we would 'observe net regeneration of N03- inour incubation experiments. These observations wereentirely serendipitous and indicative of intense nitrifi-cation. Net regeneration of N03- at 18 and 270/00in thepresent investigation was rapid and higher than nitrifi-cation (N02- + N03- production) rates reported fromthe Rhone River, France «0.01 to 0.18 JIM hot; Feliatra& Bianchi 1993), the Tamar River estuary, UK «0.2 pMh-1; Owens 1986), and Narragansett Bay, RhodeIsland, USA « 0.46 pM h-I; Berounsky &Nixon 1990).A comparison of net N03- regeneration to net NH4+

and N02- fluxes at 18 and 270/00(Table 5) further indi-

Table 6. Initial and final concentrations (:t 1 SD) of solublereactive phosphorus (SRP) during experimental incubations

of Mississippi River plume waters, May 1992

cated that nitrification was a significant component ofthe plume N cycle during the period investigated.

Fox et al. (1987), noting elevated N02-, N03- andN20 concentrations in the lower salinity (2 to 16%0)region of the Southwest Pass plume, concluded thatnitrification occurred in the Mississippi River upstreamfrom their sampling locations. Net regeneration ofN02- at 00/00in the present investigation also suggestednitrification occurred in the fresh waters of the delta.

Although elevated concentrations of N02- wereobserved at 100/00,the lack of significant net fluxes ofN02- and N03- suggested nitrification was negligibleat this station. The high rates of net N03- regenerationmeasured at 18 and 270/00,however, indicated thedevelopment of intense nitrification at intermediateplume salinities. The above suggested that nitrificationalong the plume salinity gradient during the presentinvestigation was partitioned between nitrification inthe Mississippi River, 'supported by riverine sources ofNH4+, and nitrification within the plume supportedby the in situ regeneration of NH4+ at intermediateplume salinities.

Although high rates of nitrification were observed atintermediate salinities, NH4+ concentrations were low« 2.5 JIM) across the entire plume salinity gradient.The latter indicated that nitrification at intermediate

salinities was supported by the rapid regeneration ofNH4+ within the plume. Dark net NH4+ regenerationrates (Table 5) in the present investigation were similarto dark NH/ regeneration rates (0.05 to 0.22 JIM h-I)reported from the plume by Gardner et al. (1994) and15NH4+ regeneration rates «0.05 to >4.5 JIM h-I)obtained under simulated in situ light conditions(Dortch et al. 1992a, Cotner & Gardner 1993). The dis-tribution of NH4+ regeneration reported by the aboveauthors and observed in the present investigation alsoindicated that NH4+ regeneration within the plumeis often highest at intermediate salinities where weobserved the highest rates of nitrification.

To examine the hypothesis that the rapid remineral-ization of organic N supported nitrification at inter-mediate plume salinities, we compared the massbalance of DIN at the beginning and end of eachexperimental incubation (Table 7). The mass balancedata indicated a net increase in DIN.at all stations and

Table 7. Mass balance of DIN during experimental incubationof Mississippi River plume water during May 1992

Table 5. Net NH4+, N02- and N03- flux rates from the South-west Pass plume of the Mississippi River, May 4-13, 1992.Positive values indicate net production, negative values

indicate net uptake

Salinity Net flux rate r2 p n Incubation time(%0) (J.1Mh-I) (h)

NH4+0 + 0.27 0.74 <0.01 15 12

10 + 0.40 0.77 <0.01 9 618 + 0.43 - 0.04. 3 227 - 0.06 0.44 <0.01 19 12

N02-0 + 0.06 0.56 <0.01 15 12

10 _ 0.03 ns 0.17 0.18 12 918 _ 0.02 ns 0.15 0.29 9 427 + 0.01 ns 0.28 0.14 9 4

N03-0 -0.22 0.74 <0.01 9 6

10 _ 0.03ns 0.10 0.35 12 1218 + 0.37 0.50 <0.01 12 927 + 0.56 0.95 <0.01 15 9

nSNot significant (p > 0.05).Paired t-test, initial and 2 h concentrations

Salinity Initial SRP Final SRP Incubation(0/00) concentration concentration time

(J.1M) (J.1M) (h)

0 1.13 :t 0.47 1.06 :t 0.09 1210 0.60:t 0.15 0.42:t 0.19 1218 0.28 :t 0.03 0.22:t 0.02 1227 0.29 :t 0.05 0.22 :t 0.01 12

Salinity Initial DIN Final DIN t:.DIN t:.DIN h-I(%0) (J.1MN) (J.1MN) (J.1MN) (J.1MNh-l)

0 116.7 118.7 + 2.0 + 0.1710 66.0 67.6 + 1.6 + 0.1318 51.3 54.7 + 3.4 + 0.3827 18.2 23.1 + 4.9 + 0.54

Pakulski et al.: Nutrient cycling in the Mississippi River plume 213

(Bronk & Glibert 1993). It isnot unreasonable, therefore, tohypothesize that the mineral-ization of urea could contribute

to the NH4+ regeneration nec-essary to support the observednitrification rates in the plume.Thus, within the limitations of

our assumptions and the vari-ous analytical methods usedto estimate our N fluxes, it

appeared that remineralization of plume-derivedorganic N would be sufficient to support the high ratesof nitrification we observed in these waters.

We hypothesize a sequence whereby the mineraliza-tion of particulate organic N (paN) derived from pri-mary production at intermediate plume salinitiesresults in the regeneration of NH/ supporting nitrifica-tion. This hypothesis is similar in many respects to aconceptual model of plume bacterial DON remineral-ization presented by Cotner & Gardner (1993). In theMississippi River plume, increasingly favorable lightregimes and high concentrations of riverine DIN stim-ulate photosynthesis and the production of paN at

intermediate pl~me salinities (Lohrenz et al~~Lopez-Veneroni & Cifuentes 1992). At intermediateand higher salinities, DIN concentrations decline whileDON concentrations increase dramatically (Lopez-Veneroni & Cifuentes 1992). The above suggests thatmuch of the riverine N03- assimilated into paN byphotoautotrophs at intermediate salinities is subse-quently transformed into DON.

Amino acid uptake rates measured during 15NH4+

regeneration experiments (Cotner & Gardner 1993,Gardner et al. 1993) indicate that bacterial remineral-ization of DON contributes to the regeneration of NH/in the plume. Size-fractionation experiments, however,indicate that much of the NH4+ regeneration at inter-mediate plume salinities occurs in the > 1.0 J.lmsizefraction (Gardner et al. 1994) suggesting that themesozooplankton and other larger organisms alsocontribute to NH4+ regeneration within the plume.Mesozooplankton are present in high 'concentrations(100 to > 1000 I-I) at intermediate plume salinities(Dagg & Whitledge 1991) and consume a substantialportion of plume primary production in the late springand summer months (Dagg & Ortner 1992, Fahnenstielet al. 1992). Mesozooplankton may contribute directlyto the regeneration of NH4+ though excretion, or indi-rectly through the release of urea and other forms ofDON (Corner & Newell 1967, Lampert 1978, Vidal &Whitledge 1982, Jumars et al. 1989). Bacterial reminer-alization of DON and excretion of NH4+ by mesozoo-plankton may thus result in the rapid regenerationof NH/ at intermediate plume salinities. Cotner &

Table 8. Comparison of total community DIN fluxes (NH4. + N02- + N03 -) to N regener-ation estimated from C remineralization at 18 and 27%0.Nitrogen remineralization rates

were estimated from DIC production rates assuming Redfield C: N ratios

Salinity(%0)

Community DIN flux(pM h-I)

N remlneralization Difference(C: N =6.625) (DIN flux - N remineralization)

(pM h-1) (pM h-1)

1827

0.440.13

0.380.50

0.820.63

thus a net transformation of organic N (particulate ordissolved) to DIN. The high rates of net DIN increaseobserved at 18 and 27 % were consistent with the

hypothesis that the rapid remineralization of plume-derived organic N supported nitrification during theperiod investigated.

We also compared community DIN fluxes (the sum ofnet NH4+, N02- and N03- flux rates) to N remineraliza-tion rates derived from our estimates of DIC productionat 18 and 27%0 (Table 8). These latter estimatesassumed the mineralization of phytoplankton-derivedorganic matter exhibiting Redfield (6.6) C: N ratios.Estimates of N remineralization derived from our DIC

regeneration rates at 18 and 27%0 were 2- and 5-foldlower, respectively, than measured community DiN'fluxes at these 2 stations (Table 8). These comparisonsindicated that N03- regeneration at 18 and 27%0 wasnot supported solely by the remineralization of organicmatter exhibiting Redfield C:N ratios.

We consider below the potential contribution of ureamineralization to our N fluxes. Urea possesses a C: Nratio of 0.5 and is an end product of N metabolism inmany higher organisms including copepods (Corner &Newell 1967). It is generally assumed that urea isassimilated primarily by phytoplankton (McCarthy1972). Urea-hydrolyzing bacteria, however, have beeniSolated from coastal waters (ZoBell & Feltham 1935).

There is no information available regarding typicalconcentrations or flux rates of urea in the MississippiRiver plume. Dissolved organic nitrogen (DON) con-centrations in the plume, however, vary from 12 to50 J.lM (Lopez-Veneroni & Cifuentes 1992). Assumingthat urea comprises 5% of DON (Remsen 1971), weestimate that urea concentrations in the MississippiRiver plume may be on the order of 0.6 to 2.5 pMurea-No The latter estimates are well within the rangeof urea concentrations (0.5 to 5 pM urea-N) observedin coastal waters (McCarthy 1970, Remsen 1971,Kauffman et al. 1983, Kristiansen 1983). Urea mineral-ization rates required to balance our DIN flux rates at18 and 270/00would be equivalent to 0.38 and 0.50 JlMurea-N h-I, respectively (Table 8). The latter estimatesare similar in magnitude to 15N_urea uptake rates(0.10 to 0.29 J.lM h-I) measured in Chesapeake Bay

214 Mar. Ecol. Prog. Ser. 117: 207-218,1995

Gardner (1993) have further suggested that microbialprocesses at intermediate plume salinities may regen-erate NH/ at rates exceeding phytoplankton uptake.The latter is consistent with the observation byDortch et al. (1992a) that NH/ regeneration in thelight exceeds NH4+ uptake over much of the plumesalinity gradient. Ammonium regeneration in excess ofphytoplankton demand may consequently stimulatethe development of nitrification at intermediatesalinities.

Distribution of nitrification in the Mississippi Riverand Rhone River plumes

The discharge of the Rhone River into the northernMediterranean Sea results in the formation of a

shallow unconfined estuarine plume (Kirchman et al.1989) similar to the plume originating from the Missis-sippi River delta. The distribution of DIN concentra-tions and nitrification rates across the Rhone River

plume salinity gradient (Feliatra & Bianchi 1993),however, differ in many respects from those observedin the Mississippi River plume during the present in-vestigation. Concentrations oTNHT(9:B3 pM);-N02-(3.85 pM), and N03- (112.8 pM) were highest in theRhone River and decreased with increasing salinity(Feliatra & Bianchi 1993). Highest rates of nitrificationwere also observed in the Rhone River and declined

with increasing salinities. In the present investigation,only N03- exhibited maximal concentrations in theMississippi River and the highest rates of nitrificationoccurred at intermediate salinities. Comparison ofthese 2 plume systems suggests that nitrification in theRhone River plume was supported by NH4+ originatingfrom the Rhone River while nitrification in the Missis-

sippi River plume was associated with in situ NH4+regeneration at intermediate salinities.

Bacterial metabolism and plume communityO2consumption

Assuming nitrification consumes 2 mol O2 per moleof NH4+ oxidized to N03 -, O2 consumption due to nitri-fication at 18 and 27%0 was equivalent to 0.74 and1.12 pM O2 h-I (Table 9). The latter rates were similarto the difference between O2 consumption due tocarbon remineralization (DIC production) and totalcommunity O2 consumption at these stations (Table 9).Thus within the limitations of our estimates of O2consumption and N03 - regeneration at these stations,nitrification accounted for a substantial portion ofcommunity O2 consumption at intermediate plumesalinities.

Heterotrophic bacteria are generally considered tobe the principle consumers of O2 in marine ecosystems(Williams 1981, 1984, Hopkinson et al. 1989, Griffith etal. 1990). This generalization, however, may not holdtrue for hypereutrophic systems such as the MississippiRiver plume. Jensen et al. (1990) reported that organ-isms other than heterotrophic bacteria were responsi-ble for> 50 % of community O2consumption in a highlyeutrophic region of. a shallow Danish estuary. In theMississippi River plume, Chin-Leo & Benner (1992)reported that organisms in the > 1.0 pm size fractionaccounted for 44 to 68 % of community O2 consumptionduring the summer, suggesting that larger phytoplank-ton and zooplankton were responsible for -50 % ofcommunity O2 consumption during this period.

We estimated the contribution of nitrifying bacteria,heterotrophic bacteria and other organisms (zoo-plankton, protozoa and photoautotrophs includingcyanobacteria) to community O2 consumption at 18 and27%0. Total community O2 consumption at 18%0 was3.65 pM h-I of which 0.74 pM h-I (Table 9) was attrib-utable to nitrification. Assuming a bacterial growthefficiency of 24 % (Chin-Leo & Benner 1992) and arespiration quotient (RQ) of 1.0, O2 consumption byheterotrophic bacteria at 180/00was estimated to be1.30 pM h-I (Table 3). Oxygen consumption by otherorganisms (1.70 pM h-I) was estimated as the differ-ence between total community O2 consumption andbacterial (heterotrophs + nitrifiers) O2 consumption.Nitrifying bacteria, heterotrophic bacteria and otherorganisms thus accounted for 20, 36 and 44 %, respec-tively, of community O2 consumption at 180/00.Similarestimates indicated that nitrifying bacteria, hetero-trophic bacteria and other organisms contributed 64,9 and 27 %, respectively, of community O2 consump-tion at 27%0. The above indicated that heterotrophicbacteria were not the principal consumers of O2 atintermediate plume salinities during the periodinvestigated.

We could not evaluate' the relative contribution of

autotrophs and heterotrophs to the above estimates ofO2 consumption not accounted for by heterotrophicand nitrifying bacteria. Packard (1979) and Iriarte et al.(1991), however, have suggested that autotrophicrespiration may dominate community respirationwhere high concentrations of phytoplankton biomassare present. Previous investigations in the MississippiRiver plume (Lohrenz et al. 1990, Dagg & Whitledge1991, Dortch et al. 1992a, Hitchcock & Whitledge1992) have indicated that concentrations of phyto-plankton biomass are extremely high (10 to 45 J.Igchi I-I) at intermediate salinities. Cyanob.acteiia arealso important members of the Mississippi River plumemicrobial community and are present in con centra-

-tions (-105 cells ml-I similar to heterotrophic bacteria

----

Pakulski et al.: Nutrient cycling in the Mississippi River plume 215

Table 9. Community O2 consumption, DIC production, O2 consumption not accounted for by carbon remineralization ('excess' O2consumption =community O2 consumption minus community DIC production, assuming an RQ of 1) , O2 consumption due tonitrification (assumes 2 mol O2consumed per mol NH4+ oxidized to NO)- and O2consumption due to nitrification as a percentage

of total community O2 consumption at 18 and 27%0)

(Dortch 1994). Based on our estimates of O2 consump-tion by the plume community at 18 and 27%0, wehypothesize that respiration by photoautotrophs is animportant component of community O2 consumption inregions of high phytoplankton biomass.

Implications of nitrification for estimates of plumebacterial production and growth efficiencies

Our estimates of bacterial production were based onthe incorporation of leucine by heterotrophic bacteria.During the period investigated, however, nitrificationIdl~:; iJldicdl~d lhdt nitrifyiIrg-bacteriawere- aIso--animportant component of the plume bacterial commu-nity. The ability of nitrifying bacteria to incorporateextracellular leucine has, to our knowledge, not beeninvestigated. Nitrifying bacteria do not incorporate[3HI-thymidine (Johnstone & Jones 1989) and it isunlikely, therefore, that they incorporate [3HI-Ieucine.Consequently, leucine incorporation rates may notmeasure the contribution of nitrifying or other auto-trophic bacteria (such as cyanobacteria) to total plumebacterial production.

Chin-Leo & Benner (1992) used leucine incorpora-tion and O2 consumption rates obtained from 1.0 pmfiltered water samples to empirically estimate plumebacterial growth efficiencies. In the present investiga-tion, we used the mean value of bacterial growth effi-ciency (24 %) reported by Chin-Leo & Benner (1992) toestimate heterotrophic bacterial respiration. The lattervalue is at the low end of the range of bacterial growthefficiencies reported in the literature (20 to 80 %j ct.Bjernsen 1986). Growth efficiencies of heterotrophicbacteria in the Mississippi River plume, however, arevariable (9 to 42 %) across the plume salinity gradientand between seasons (Chin-Leo & Benner 1992). Ifnitrification contributed to O2 consumption in theexperiments conducted by Chin-Leo & Benner (1992)to estimate bacterial growth efficiencies in the Missis-sippi River plume, heterotrophic bacterial growth effi-ciencies would be underestimated. The use of higherbacterial growth efficiencies in, our calculations, how-

ever, would decrease our estimates of heterotrophicbacterial O2 consumption and further diminish the esti-mated contribution of, heterotrophic bacteria to totalcommunity O2 metabolism.

Leucine incorporation rates often increased dramati-cally over the course of the incubations (Fig. 1). At10%0,community O2consumption and NH4+ regeaera-tion were linear (r =0.88, P < 0.01 for both) over thecourse of the incubation whereas leucine incorporationrates increased nearly 2-fold. Similar observations forcommunity O2 consumption and leucine incorporationrates have been reported by Pomeroy et al. (1994)and Biddanda et al. (1994). The mechanism(s) respon-sible fO! these obsenations-remain obscttre;-The- in ---

creases in leucine incorporation rates observed in thepresent investigation indicated that some portionof the heterotrophic bacterial community at 10%0 re-sponded quickly to confinement, perhaps through in-creases in growth efficiency. Changes in leucine incor-poration rates at 10%0, however, were not reflected inchanges in community O2consumption or NH4+ regen-eration over the course of this particular experiment.

Influence of Mississippi River discharge on plumebacterial production and community respiration

Plume heterotrophic bacterial production and O2consumption rates in the present investigation were

2- to 4-fold greater t~an values measured in Februaryor July (Benner et al. 1992, Chin-Leo & Benner 1992,Gardner et al. 1994). Higher springtime rates of plumeheterotrophic bacterial production and community O2consumption suggested that these pFocesses may beinfluenced by higher seasonal discharge from the Mis-sissippi River. By comparing bacterial production inthe Mississippi River to bacterial production at inter-mediate plume salinities, Chin-Leo & Benner (1992)concluded toat plume bacterial production was sup-ported primarily by river-borne substrates in the win-ter and by phytoplankton-derived substrates in sum-mer. Applying th'e model employed by Chin-Leo &Benner (1992) to bacterial production data from the

Salinity Community O2 Community DIC 'Excess' O2 O2 consumption due O2 consumption due(%0) consumption production consumption to nitrification to nitrification as %

(pM h-I) (pM h-l) (pM h-I) (pM h-I) (% community O2consumption)

18 3.65 3.00 0.65 0.74 2027 1.74 0.85 0.89 1.12 64

216 Mar. Ecol. Prog. Ser. 117: 207-218, 1995

present investigation, we estimate that phytoplankton-derived substrates supported 82 % of heterotrophicbacterial production at intermediate plume salinities.The latter suggests that bacterial production observedin the present study was stimulated by enhanced pri-mary production at intermediate salinities rather thanby input of organic matter from the river. The distribu-tion of O2 consumption and DIC production also indi-cated that community respiratory activity was sup-ported by autochthonous production of organic matterat intermediate salinities.

Nutrient limitation, microbial N-cyling and 'new'production in the Mississippi River plume

While the supply of N is generally assumed to limitbiological production in many marine systems (e.g.Ryther & Dunstan 1971) the extent of either P- or N-limitation in coastal marine environments remains

unclear (Hecky & Kilham 1988, Hecky et al. 1993). Theresults of P-addition experiments (Chin-Leo & Benner1992) and the rapid turnover of inorganic 32pin Missis-sippi River plume waters (Ammerman 1992) suggested-tbe-potential-fof--P-limitation--in-tBe-s\:IHHl1eF-ilBd falh-The high (>60:1) DIN:SRP ratios observed throughoutthe plume in the present investigation further sug-gested the potential for P-limitation of plume bacterialand phytoplankton production in the spring. Whileinferring nutrient limitation solely from external nutri-ent ratios is speculative, the above suggests that P maypotentially limit bacterial and primary production inthe Mississippi River plume during much of the year.

Turner & Rabalais (1991) hypothesized that rapidrecycling of N within the plume would amplify theeffects of N loading from the Mississippi River on theinner Gulf shelf. The lower molar N03 -: NH4+ ratios atsalinities 27 and 360/00indicated the increasing impor-tance of regenerated DIN at higher plume salinities.The high rates of net N03 - regeneration observed at18 and 270/00in the present investigation, however,indicated that some portion of the N03- present at'intermediate salinities was regenerated from nitrifica-tion and thus not entirely 'new' (i.e. based on theassimilation of riverine or upwelled N03 -) as originallydefined by Dugdale & Goering (1967). Ward et al.(1989) have suggested that the assimilation of N03-regenerated via nitrification in the nitricline of theCalifornia Bight obscured the distinction between newand regenerated N-based primary production in thesewaters. The high rates of N03- regeneration measuredat intermediate salinities in the present investigationsuggested that measuring 'new' and 'regenerated' pri-mary production based solely on the uptake N03 - maybe unreliable in coastal marine ecosystems as well.

Nitrification in the Mississippi River plume:implications for the development of hypoxia

on the inner Louisiana shelf

Evidence from the present investigation indicatedthat nitrification was an important mechanism con-tributing to O2 consumption in the Mississippi Riverplume. Our data further suggested that the high ratesof nitrification observed in the present investigationwere supported by the rapid remineralization oforganic N derived from nutrient-enhanced primaryproduction at intermediate salinities. Previous research

(e.g. Lohrenz et al. 1992) has indicated that N inputsfrom the Mississippi and Atchafalaya Rivers supporthigh levels of primary production over extensive areasof the Louisiana shelf subject to the seasonal develop-ment of hypoxia. Much of the organic N derived fromnutrient-enhanced primary production on the innerLouisiana shelf may be remineralized to NH4+ indeeper waters during periods of enhanced water-column stratification in the summer months. We specu-late the latter may in turn stimulate nitrification which,in conjunction with O2 consumption resulting from themineralization of organic C, contributes to the forma-

--tien--of-hypoxia-ffi--thesiHVater~ .

Acknowledgements. This research was supported by theCoastal Ocean Program Office of the NOAA through grantNA 90AA-D-SG689 to the Texas Sea Grant Program. Theauthors thank the following: D. Shorman for the nutrientanalyses, M. Lansing for assistance with sampling and DICanalyses, and the crew of the RV 'Longhorn' for theirassistance in sampling. University of Texas at Austin MarineScience Institute publication no. 933.

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This article was presented by S. Y. Newell (Senior EditorialAdvisor), Sapelo Island, Georgia. USA

Manuscript first received: April 19, 1994Revised version accepted: August 31, 1994