Hindawi Publishing CorporationJournal of EcosystemsVolume 2013 Article ID 316709 9 pageshttpdxdoiorg1011552013316709
Research ArticleShort-Term Photochemical and Biological Unreactivity ofMacrophyte-Derived Dissolved Organic Matter in a SubtropicalShallow Lake
Ng Haig They1 David Motta Marques2 Rafael Siqueira Souza2
and Luacutecia Ribeiro Rodrigues2
1 Programa de Pos-Graduacao em Ecologia Instituto de Pesquisas Hidraulicas Universidade Federal do Rio Grande do Sul91501970 Porto Alegre RS Brazil
2 Instituto de Pesquisas Hidraulicas Universidade Federal do Rio Grande do Sul 91501970 Porto Alegre RS Brazil
Correspondence should be addressed to Ng Haig They haigtheygmailcom
Received 7 May 2013 Revised 5 July 2013 Accepted 7 July 2013
Academic Editor Wen-Cheng Liu
Copyright copy 2013 Ng Haig They et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Macrophytes have been associated with low bacterial metabolism in the littoral zones of lake Mangueira but an explanationfor this pattern is largely unknown In this study macrophyte-derived DOM was incubated in situ for the measurement of theeffect of grazers bacteria and light on its degradation in three experiments The water was separated in bulk bacterial andcontrol (+HgCl
2) fractions and exposed to or hidden from sunlight for 120 h Unchange in bacterial variables in the bulk fraction
suggested a combined control of radiation and grazing on bacteria Light treatment increased bacterial density but not biomass andbiovolume while bacterial density decreased in the dark Significant fading of water color in the bacterial fraction only occurredafter light exposure indicating a complementary pathway of light and bacteria DOC and the Abs250 365 ratio did not change withincubation indicating no net change of DOC pool and reactivity Due to continuous carbon loading frommacrophytes and lowUVirradiance the very low rates of DOM degradation provide the mechanistic explanation for the observed impacts of macrophytesin lakersquos carbon metabolism in littoral zones
1 Introduction
Macrophytes are important sources of carbon to the littoralzones of lakes and these plants directly and indirectly (viasupport of epiphytes) contribute higher amounts of dissolvedorganic carbon (DOC) than do algal sources [1] They arethe main sustainers of bacterial production in some systems(eg [2]) but knowledge of their impact on the entire lakemetabolism is still relatively sparse [1 2]
Loading of macrophyte-derived carbon in littoral zonescan create within-lake patchiness in the quantity and qualityof organic carbon thus causing differences in the com-position of bacterial assemblages [3] Patchy utilization ofheterogeneous DOC by compositionally or adaptively dif-ferent bacterial communities could affect the entire lakemetabolism because bacterial secondary production and
respiration can be affected by the consumption of either highor low-molecular-weight compounds [4]
DOC derived from macrophytes is composed mostlyof aromatic and aliphatic polymer-like compounds of highmolecular weight [5] and hence presumably refractory andhence highly unreactive (ie with low capacity to undergochemical reaction specially oxidation) to bacterial consump-tion This requires bacterial assemblages that inhabit humiclakes to use a different more energy-expensivemechanism toexploit these compounds [1 6] Moreover this type of DOCis qualitatively deficient with low nitrogen and phosphoruscontents [7]
Once they enter the water high-molecular-weight com-pounds can follow two degradation paths they can be trans-formed into lower-weight compounds by microbial decom-position [3] or by photodegradation a process that enhances
2 Journal of Ecosystems
bacterial growth because it releases low-molecular-weightcompounds making them directly available to bacteria [8ndash12] These two routes bacterial and photo-degradationcan also be complementary that is bacteria and light actin concert decomposing different fractions of the DOCpool (autochthonous low-molecular-weight DOC mainlyby bacteria and allochthonous high-molecular-weight DOCmainly by light) [13] Additionally bacterivores (flagellatesand ciliates) can enhance decomposition rates [14] becauseof enhanced bacterial production on protist excretions [15]
Evidence is accumulating that bacterial metabolism canbe lower in the presence of macrophytes at some instancesRooney and Kalff [16] surveyed nine lakes with differentpercentages of macrophyte coverage and found a significantdecrease in the bacterial respiration rate with increasingmacrophyte coverage In southern Brazil a study in shallowcoastal lakes found that bacterial metabolism and biovolumewere frequently lower in littoral than pelagic zones and asso-ciated this with the influence of macrophytes [17] Thereforethe environmental metabolic pathways of the organic matterthat originated from macrophytes are still not understood inthese ecosystems
Previous studies [17] have pointed that the bacterialdegradation of macrophyte-derived carbon is lower in thelittoral zone but a mechanistic explanation is still lackingWe hypothesize that since macrophytes contribute with highloadings of refractory material [5] the photo-degradationmay play a major role in breaking down DOC in littoralzones In the present study we designed three independentexperiments carried out simultaneously to test the role ofbacteria bacteria under grazing pressure and light in thedegradation of DOC derived from macrophytes entering thelittoral zone of the lakeMangueira a large subtropical shallowlake
2 Materials and Methods
21 Study Site Lake Mangueira (80 800 ha state of RioGrande do Sul Southern Brazil) is a large shallow coastalsubtropical lake (Figure 1) Extensive belts of wetlands arelocated north and south of the lake The experiments werecarried out in situ with water from a channel that drainsa large wetland belt into the southernmost part of the lake(coordinates minus33∘311015840454810158401015840 minus53∘71015840566410158401015840) This channel hashigh coverage of submersed macrophytes and the waterentering the lake is highly colored compared to the adjacentwaters in the pelagic zone Hence we assume that mostof the DOC found in this water is mainly originated bymacrophytesThis lake has been investigated for phytoplank-ton zooplankton benthos and ecological modelling [18 19]within theBrazilian Long-TermEcological ResearchProgram(PELD-Taim) and part of the lake lies within a conservationunit (ESEC-Taim) Only one study concerning heterotrophicbacteria in Lake Mangueira has been published so far [17]
The National Institute of Meteorology (INMET) stationlocated at Santa Vitoria do Palmar (minus33∘3110158400010158401015840 minus53∘21101584000Altitude 2401m) provided meteorological data such asmean air temperature (AT) No noticeable precipitation was
recorded during the experiment Global Radiation (GR)(the sum of the radiation that come directly from the sunplus diffused radiation) was estimated by satellite [20] sup-ported by the Brazilian National Institute of Space Research(CPTEC-INPE) Total radiation (accumulated global radia-tion) during the experiment was calculated from the dailymean flux radiation times the hours of sunshine (providedby INMET) Accumulated UV radiation was estimated bymultiplying the total radiation by 23 times 10minus3 which is theproportion of UV radiation in total radiation The dailyUV-index (1 unit = 25mWmminus2) during the experiment wasprovided by INPE (National Institute of Space Researchlthttpwwwinpebrgt)
22 Channel Water Characterization All the following vari-ables were measured in triplicates in the channel waterto characterize the experimental conditions alkalinity wasmeasured through Gran titration method and the pH witha potentiometer (TecnoponMPA 210p Piracicaba SP Brazil)[21] Total solids (TS) weremeasured gravimetrically throughwater evaporation in porcelain dishes [22] Total nitrogen(TN) and total phosphorus (TP) weremeasured by colorime-try [23] Dissolved organic carbon (DOC) was determined bya TOC Analyzer (Shimadzu VCPH Columbia MD USA)as the fraction that passed through a 450∘C precombustedglass fiber filter (Macherey-Nagel GF6 06 120583mmean particleretention size) Absorbance at 430 nm was measured as anestimate of water color and the proportion of low- to high-molecular-weight substances as the ratio Abs250 365 [824] To determine the total density of ciliates 50mL of thesample was fixed with lugol (91 volvol) and samples werestored in the dark and cold Total samples were counted insedimentation chambers under an inverted microscope andreported as individuals times104mminus3 [21] Bacterial productionrespiration and BGE were measured the same way as for theexperiment (see below)
Alkalinity pH Abs430 and Abs250365 were measuredimmediately in the field laboratory Samples for TS TN andTPwere immediately frozen in 1 L polyethylene bottles DOCsampleswere collected in 30mLprecombusted (450∘C for 1 h)amber glass bottles and acidified with H
3PO4
minus3
23 Experimental Procedure Surface water was collected inthe channel with the help of a plastic bucket and broughtto the field laboratory We set up three independent andsimultaneous experiments we separated the water into bulkbacterial and control fractions and measured variables intriplicates in the initial condition and exposed (light) ornot (dark) to sunlight totaling 27 incubation bags Thebulk fraction with no filtration was used to evaluate theeffect of grazing the bacterial fraction was obtained afterfiltration on MN 640d Macherey-Nagel paper filters (meanretention size of 20 to 40120583m) in order to exclude mostbacterivores This unusually permissive retention size wasthe best found to isolate bacteria and retain all metazoansciliates and most flagellates in many previous pilots withdifferent filters (including less permissive ones) ([17] and thisstudy) The control was obtained after the same filtration as
Journal of Ecosystems 3
minus100 minus80 minus60 minus40
minus50
minus40
minus30
minus20
minus10
0
10
Brazil
minus326
minus330
minus334
minus530minus535 minus525
Figure 1 Sampling location the large subtropical shallow LakeMangueira in southern BrazilThe experiment was carried out in its southernpart
the bacterial fraction and addition of the inhibitor HgCl2
(04 120583gmLminus1 final concentration) in order to isolate thephoto-degradation effectThis inhibitor has no effect on pho-toreactivity (the capacity of undergoing oxidation by light)of DOM or UV-visible absorption spectra [25] PolyethyleneWhirl-Pak bags (Nasco Pittsburgh USA) of 500mL capacity(115times23 cm) were filled with 350mL of each fractionThesebags are UV-transparent and did not present differencesin bacterial activity in comparison with quartz containersduring sunlight exposition [26] Apart from the initial bags(9 bags) which were sampled just before the beginning of theincubation 9 bags were protected from sunlight by double-wrapping with aluminum foil the 9 remaining bags wereexposed to natural sunlight Because of the headspace in thebags they floated at the surface and there was no water layerat the upper side of the bags hence we assumed a negligibleabsorbance of UV by the water The bags were randomlydeployed on a plastic tray (1m2times 10 cm high open on theupper side) made of coarse-mesh shade cloth (3 cm times 1 cm)tied to a floating PVC pipe square also 1m2 The tray wasanchored in an unvegetated littoral zone at another pointin the lake (asymp05m depth) away from the sampling pointfor 120 h Farjalla et al [11] conducted an experiment withmacrophyte leachates and bacterial decomposition and foundan endpoint of 96 h of incubation when bacterial numbersreached a plateau related to the total amount of availablegrowth substrate In our study we extended this period byone day because of lack of prior knowledge of what endpointwould apply to our case
24 Experimental Variables The collection and preservationof samplings were carried according to Haig-They [17]Samples for cell counting biovolume and biomass were fixed
in 4 formaldehyde (vol vol) in polyethylene bottles in thefield and stored in the dark at 4∘C until analysis Samplesof the bulk fraction were prefiltered on quantitative paperMN 640d Macherey-Nagel mean mesh size 20 to 40 120583min order to exclude organisms other than bacteria In thelaboratory 2mL of each sample was filtered (lt50 kPa) ina Vacuum Manifold Filtration Tower (Millipore BillericaMA) with 1 mL of Milli-Q water (02120583m filtered) to improvecell dispersion Cells were concentrated on 02 120583m blackpolycarbonate membranes (GE) Approximately 1mL of 10(weight vol) acridine orange stain was added to the filters for5min They were then washed with Milli-Q water and air-dried Filters weremounted on slides withmineral oil (Nujol)and photographed within 3 days A total of 10 images werecaptured per filter and image processing was undertakenon six of them with the help of an image grab systemWe employed a MOTIC 5000 cooled camera coupled to anOlympus IX70 inverted epifluorescence microscope (CenterValley PA USA) Image capture (MOTIC Image 32) andprocessing followed Massana et al [27] with the help of theFreeware Image Tool (v300) The processed and binarizedimages were then used for cell counts and determinationof dimensions Cell density was determined from the meannumber of cells in 6 images and the cell-density equationof Kepner and Pratt [28] Bacterial biovolume (120583mminus3) wasassigned to each cell according to the morphotype [27]utilizing the cell dimensions and morphotypes classificationprovided by CMEIASImage Tool (127) [29] The meanbiovolume per cell was calculated as the mean of all cellsin all images Bacterial biomass (pg C cellminus1) was calculatedemploying an allometric function of biovolume [30] and themean from all cells in all images was multiplied by the celldensity to yield the bacterial carbon concentration of the
4 Journal of Ecosystems
sample (ng C mLminus1) Bacterial production rates (BP) wereestimated through the method of (L[45-3H]) radiolabeledleucine microcentrifugation Incubations for bacterial pro-ductionwere carried out in Eppendorf vials (2mL) and lasted30min inside a tray filledwith lakewater at room temperature(15-16∘C) Radioactivity counts were made after addition of1mL of scintillation liquid (Optiphase HiSafe III Wallac)in a Rack Beta Liquid Scintillation Counter (LKB Wallac1209 Massachusetts USA) for 180 s twice The calculationsassumed the isotopic dilution to be equal to 2 the molar ratioof leucine in the protein pool to be 0073 and the ratio ofcarbonprotein contents to be 0086 [31 32]
The bacterial respiration in the fractions (bulk bacterialand control) was estimated as the rate of oxygen consump-tion through the Winkler method [33] with colorimetricreading [34] This method was conducted separately througha parallel incubation of the fractions in glass BOD bottles of100mL capacity Incubation was done in the dark (double-wrapping the bottles with aluminum foil) with a total of 18bottles (9 initial bottles and 9 final bottles three triplicatesof each of the three fractions) and then only comparisonsamong fractions could be made The bottles were tied bytheir necks to another floating PVC pipe square that wasanchored next to the incubating bags For calculations eachfinal value of O
2was subtracted from the mean of the three
initial bottles and divided by the incubation time assumingconstant respiration rate over time The molar conversionfactor between carbon and oxygen was assumed to be equalto 10 [35]
Bacterial GrowthEfficiency (BGE)was computed accord-ing to equation (I) of del Giorgio et al [36] (I) BGE =(BP)(BP + BR) where BP is the bacterial production rateand BR is the bacterial respiration rate Because respirationwas recorded only for the three fractions also BGE couldonly be calculated and compared among fractions For thecalculations in order to match the results from respirationwe employed the mean of the bacterial production ratebetween the initial and final incubations
Ciliates Abs430 and Abs250 365 were measured thesame way as for field samples [8 21 24]
25 Statistical Treatment Differences in the variables (bacte-rial density biovolume biomass production ciliate densityDOC Abs 430 nm and Abs250 365 nm ratio) among initiallight and dark treatments in each fraction were tested withone-way ANOVA using R 2131 [37] Significant differencesrevealed by ANOVA were tested by Tukeyrsquos a posteriori testIncreases or decreases refer to the light and dark treatmentsin relation to the respective initial condition Bacterial respi-ration and BGE were tested for differences among fractionsand field condition through ANOVA and Tukeyrsquos test
3 Results
The main limnological variables for the experimental condi-tions can be found in Table 1The global cumulative radiation(GR) during the experiment was asymp25 455KJmminus2 Total UVradiation received was estimated to be asymp58 kJmminus2
Bacterial density did not differ among treatments in thebulk fraction it increased with light exposure and decreasedin the dark bags in the bacterial fraction In the controlfraction there was an increase in density in the dark treat-ment Bacterial biovolume did not change with treatmentsBacterial biomass also showed no differences in the bulkfraction It decreased in the dark treatment for the bacterialfraction and increased in the dark treatment in the controlfraction (Table 2)
Bacterial production was unaltered in the bulk fractionIn the bacterial fraction it decreased in the light treatmentand increased in the dark In the control fraction it increasedin the dark Bacterial respiration and BGE did not differamong the fractions (Table 2)
Comparing the bacterial production with the field con-dition only the dark control treatment showed a significantdifference (ANOVA F(920) = 2941 119875 ≪ 0001 dark controlgt Field 119875 ≪ 0001) Bacterial respiration did not differ fromthe field condition in any treatment (ANOVA F(38) = 2517119875 = 0132) BGE decreased in the bulk and bacterial fractionincubations (ANOVAF(38) = 492119875 = 00318 FieldgtBulk119875 = 0058 Field gt Bacterial 119875 = 0052)
In the bulk fraction total ciliate density changed only inthe light treatmentThe light treatment at its turn decreasedcompared to the initial treatment The dark incubation didnot differ statistically from the initial treatmentThe filtrationprocedure successfully excluded ciliates as no ciliates werefound in the bacterial and control fractions any ciliate inthe control was likely lysed by the toxic inhibitor (Table 2)No flagellates were found in the filtered samples undermicroscopic examination
DOC and Abs250 365 showed no effect of treatmentfor any fraction Abs430 (water color) was unaltered in thebulk fraction for all treatments only the bacterial fractionshowed significant color fading in the light treatment (26lower)The control light bags did not show any alterationwithtreatment but the color was less intense than in the dark bagsRates of fading in the light treatment were 00006 00014and 00012 dayminus1 absorbance units for the bulk bacterial andcontrol fractions respectively (Table 2)
4 Discussion
The littoral zones of Lake Mangueira (especially in thenorth and south) are extensively colonized by emergent andsubmersed macrophytes and these plants are expected tocontribute large amounts of organic carbon to the systemMuch of this carbon enters the lake in the form of DOMwhich we found to show little reactivity (did not undergodecomposition by bacteria or light) during short-term incu-bation under in situ conditions We were unable to detectchanges in DOC Abs250 365 and bacterial respirationalthough the water color faded in the light bags Theseresults indicated that photodegradation did occur but didnot modify carbon availability to bacteria Exposure tolight however had contrasting effects it increased bacterialdensity in the bacterial fraction suggesting a positive effectof light but decreased bacterial production in this same
Journal of Ecosystems 5
Table 1 Limnological and biological characterization of the water in the initial conditions of the experiment
1Mean air temperature for the 5 days of incubation2Mean daily flux of GR during incubation (n = 5) 1W = 1 J sminus1 For all other variables n = 33Figures in parentheses are plusmnstandard deviations4119885mean mean depth (m) AT air temperature (∘C) TS total solids (mg Lminus1) Alk alkalinity (120583Eq Lminus1) TN total nitrogen (120583g Lminus1) TP total phosphorus(120583g Lminus1) DOC dissolved organic carbon (mg Lminus1) Abs430 absorbance at 430 nm GR global radiation flux (Wmminus2) LMW low-molecular-weight substances(absorbance at 250 nm)HMW high-molecular-weight substances (absorbance at 365 nm) Abs250 365 (ratio of absorbances at 250 and 365 nm) BP bacterialproduction (120583gC L hminus1) BR bacterial respiration (120583gC L hminus1) BGE bacterial growth efficiency
treatmentfraction suggesting photoinhibition We did notexamine the effect of exposure to light on respiration but ourresults showed no difference in respiration from a previousmeasurement in the field (Table 1) The decrease in BGEwith incubation can be attributed to the increase in respi-ration (even though there was no significant differences inrespiration among fractions it impacted the BGE calculationbecause respiration tended to be higher in bacterial andcontrol fractions)
The general lack of changes in practically all variables inthe bulk fraction indicates that there may be an interactionof grazing with radiation The increase of bacterial densitywith light found in the bacterial fraction may have beenimpeded by grazing in the bulk fraction Higher densitiesof bacteria and bacterial grazers have been found in UV-irradiated DOC suggesting that photo-degradation acts as astimulus to microbial food webs [38] In our case howeverthe decrease in ciliate density with light treatment in thebulk fraction indicated a net effect of photoinhibition onciliates If they did control bacterial density it was at acost of a higher grazing rate per cell On the other side ithas been found that high concentrations of refractory DOClike in humic lakes may be associated to lower bacterialnanoflagellates and ciliates numbers and biomass and hencea less efficient functioning of the microbial loop that islower rates of DOC reincorporation to the food web [39]The density of ciliates in the initial condition (presumably afield concentration estimate) and inside the bags was low (2ndash4 orders of magnitude) when compared to values reportedfor lakes with variable trophic status where they range from19 to 100 times 106 cellsmminus3 (see compilation in Gates [40])Thissuggests that there may be a minor role of ciliates on thedecomposition rates of this refractory DOC in Mangueiralakersquos littoral zones
Unexpectedly we found enhancement in bacterial den-sity biomass and production in the dark treatment of thecontrol fraction This enhancement was first attributed tosome kind of contamination but since no ciliates werefound in these bags and contamination in the three bagssimultaneously is unlikely this result was not disregardedAlthough we were not able to explain this unexpected resultdevelopment of organisms capable of metabolizing mercuryis possible Several common bacterial genera (EscherichiaEnterobacter and Bacillus) are able to resist HgCl
2through
mercury methylation [41]This has compromised the evalua-tion of the photo-degradation alone and needs to be criticallyconsidered
An important point of our study is that we were able tofind significant fading of DOM only in the light treatmentof the bacterial fraction which suggests the existence of acomplementary pathway of dissolved organic degradation bybacteria and light [13] Farjalla et al [11] also found fading(in terms of 250 365 and 430 nm absorbance) of DOM butdecreased bacterial density and production of bacteria inUV-exposed macrophyte leachates indicating that fading itselfsays little about the bioavailability of compounds formedTheincrease in bacterial density together with the stability of theDOC and Abs250 365 suggests that the photodegradationof high-molecular-weight compounds may have producedassimilable substances for bacterial growth [9 10] but theywere readily consumed by bacteria Another important ques-tion still concerning mercury inside control bags is thatthis metal shows great affinity with organic matter alteringthe conformation of the latter [42] other bivalent heavymetals like Cu+2 Pb+2 and Cd+2 have been found to changephotochemistry of yellow substances and humic acids [43]and it is possible that mercury has had some unevaluatednegative effect on photochemical reactivity in the bags whereit was added to
Simultaneous irradiation and incubation in our studymay have exposed bacteria to continuous production ofinhibitory free radicals as suggested by the decrease inbacterial production Many studies that dealt with bacterialutilization of photochemically transformed organic carbonderived from macrophytes employed an approach of pre-treating the leachates with UV light prior to the incubationof bacterial batches [11 13 44] In many cases this priorexposure has been found to inhibit bacterial production anddecrease growth efficiency at least for a short period whichhas been attributed to the formation of hydrogen peroxideand possibly other free radicals by light [11 45] Formationof nonassimilable compounds is also possible Seitzingeret al [46] studied bacterial utilization of dissolved organicmatter in two streams and found that BP increased greatlyduring two days of incubation while DOC concentrationsignificantly decreased (45ndash50) in 12 days However 60of the compounds found in the complex mixture of organicmolecules were not consumed which Seitzinger et al [46]
6 Journal of Ecosystems
Table2Differencesb
etweeninitialcond
ition
andexpo
sition(light)or
not(dark)o
fmacroph
yte-deriv
edDOM
tosunlight
inun
filteredwater
(Bulk)filteredwater
(Bacteria
l)andCon
trol
(HgC
l 2)for120
hun
derinsitucond
ition
sinthesouthern
partof
thesubtropicalshallo
wlake
Mangueira
(sou
thernBrazil)
teste
dby
one-way
ANOVA
andTu
keyrsquos
test(m
eanplusmnsta
ndard
deviation)
Experim
ent
(I)B
ulk
(II)Ba
cterial
(III)C
ontro
lInitial
Light
Dark
Initial
Light
Dark
Initial
Light
Dark
Bacteriald
ensity
(cells10
6mLminus
1 )081plusmn016
a076plusmn050
a051plusmn041
a17
1plusmn031
a265plusmn009
b063plusmn007
c14
3plusmn013
a10
3plusmn010
a218plusmn033
b
Bacterialbiovolume
(120583m
3 )006plusmn002
a005plusmn001
a005plusmn001
a010plusmn001
a008plusmn001
a006plusmn001
a006plusmn001
a007plusmn002
a007plusmn003
a
Bacterialbiomass
(ngC
mLminus
1 )65plusmn026
a372plusmn206
a333plusmn320
a1864plusmn591
a2473plusmn277
a414plusmn045
b95
7plusmn13
1a807plusmn18
5a1649plusmn357
b
Bacterialprodu
ction
(120583gC
Lminus1 hminus1 )
012plusmn004
a028plusmn024
a015plusmn003
a015plusmn001
a002plusmn002
b014plusmn002
a003plusmn001
a003plusmn002
a077plusmn020
b
Bacterialrespiratio
n(120583gC
Lminus1 hminus1 )
NA
NA
109plusmn012
aNA
NA
228plusmn088
aNA
NA
214plusmn12
7a
BacterialG
rowth
Efficiency
NA
NA
011plusmn002
aNA
NA
007plusmn003
aNA
NA
022plusmn012
a
Ciliatedensity
(times10
4cells
mminus3 )
414plusmn18
0a18
0plusmn312
b1560plusmn178a
0plusmn0
0plusmn0
0plusmn0
0plusmn0
0plusmn0
0plusmn0
Diss
olvedOrganicCa
rbon
(mgC
Lminus1 )
1869plusmn087
a1946plusmn112
a2626plusmn78
3a2507plusmn136
a4358plusmn255
a3534plusmn204
a2090plusmn354
a3575plusmn367
a46
10plusmn175a
Abs430
(Abs
430n
m(times10minus2))
251plusmn022
a221plusmn012
a263plusmn019
a269plusmn006
a19
8plusmn014
b235plusmn020
a244plusmn016
ab18
4plusmn035
b271plusmn025
a
Abs250
365
nmratio
085plusmn009
a085plusmn002
a085plusmn004
a076plusmn007
a079plusmn018
a087plusmn002
a075plusmn005
a079plusmn025
a087plusmn006
a
Sign
ificanceo
fone-w
ayANOVA
andTu
keyrsquos
aposte
rioritestatPlt005
isindicatedwith
different
lowercase
superscriptletterswith
ineach
experim
ent(Initialversus
LightversusD
ark)Th
eexceptio
nisbacterial
respira
tionandgrow
theffi
ciencyw
here
differences
weretestedam
ongexperim
ents
NAn
otavailableb
ecause
respira
tionwas
measuredin
dark
bottlesFor
allvariables
n=3replicates
Journal of Ecosystems 7
suggested may have resulted from some inhibition factorinvolved in their utilization Contrastingly Perez et al [12]exposed natural river-water DOM to light also in a simulta-neous exposure and found bacterial enhancement with UVexposure These differences could be due to variations inorigin age and biochemical composition of the DOM [45]
The generally low bioavailability of DOC in the formof humic substances [6] indicates that a large proportionof the dissolved carbon in Lake Mangueira is refractory tobacterial consumption This Abs250 365 ratio is higher inthe littoral zone than in the pelagic zone suggesting higherproportion of low-molecular-weight (LMW Abs 250 nm)substances in the littoral zone when compared to the pelagiczone In fact this was confirmed (pelagic Abs250 mean= 003 standard deviation = 0002 F(14) = 1163 119875 lt0001 littoral zone data obtained from Table 1) In spiteof the smaller size of their molecules these substances canbe less reactive Amon and Benner [4] conducted a cross-environment experiment where they analyzed the reactivityof low-(LMW lt1 KDa) and high-molecular-weight (HMWgt1 KDa) dissolved compounds and found significant changesin BP and BR during the experiment (also five days)Unexpectedly these bacterial variables were higher in HMWsubstrates and based on these results the authors proposedthe size-reactivity continuum model that predicts that themajor path of degradation goes from large highly reactiveto small highly recalcitrant molecules If this hypothesis isbroadly applicable to many ecosystems it suggests that inLakeMangueira the compounds lixiviated by themacrophytebelt had already degraded to some extent and accumulatedas dissolved unreactive LMW compounds with very lowdegradation rates in the littoral zones
The age of the DOM subjected to bacterial degradationcan be fundamental for its availability In our experiment wefocused on the subsequent stage of macrophyte degradationthat is after bacterial and light reworking had alreadyoccurred inside the macrophyte stands Most studies thatinvestigated microbial degradation of macrophytes focusedon the early stages of degradation with exposure of coarsefragments of macrophytes to leaching For example Wehret al [47] found a 45-fold increase in bacterial productionafter 168 h of incubation of macrophyte detritus ConverselyHolm-Hansen et al [48] demonstrated very low rates ofmicrobial degradation of the emergent macrophyte Juncuseffusus only 23of the total biomass in leaf litter bagswas lostafter 268 days Enhancement of bacterial production mightbe related to the early stage of degradation given that Kuehnet al [49] employed senescent leaves in their experiment asituation more similar to ours
One possible explanation for the low photodegradationrates may be the total amount of UV accumulated during theexperiment In the study of Farjalla et al [11] macrophyteleachates were photochemically transformed with total UVenergy asymp5670KJmminus2 which was able to induce a bacterialpositive response This UV intensity is almost 100 timeshigher than that estimated for the field conditions in thepresent study (asymp58KJmminus2) One implication is that underlaboratory conditions the rates of photo-degradation may
be overestimated compared to natural conditions Anotherimportant point however is that polyethylene bags used inthis study may present a reduced rate of UV transmittance(eg 68 of UVB at 300 nm) [48] Even though the trans-mittance increases slightly towards larger wavelengths [26]this quenching may have imposed slightly reduced rates ofphoto-degradation than in the field
We attempted to determine the importance of the twomain routes of carbon degradation in aquatic systemsthat is bacterial and photo-degradation on macrophyte-derived DOM flowing into Lake Mangueira from a largewetland belt Remarkably our results showed few changesin environmental parameters related to carbon degradation(DOC Abs250 365 and respiration) even though bacterialvariables did show changes under lightdark incubationconditions (120 h) This DOM that enters the lake seemsto be highly unreactive in a short-term and may be thecause for the difference in bacterial metabolism between thelittoral and pelagic zones reported by Ng et al [17] sincethis is the main carbon source for bacteria in the littoralzone It is important to take into consideration that it wasthe first approach to the subject in this lake and was aonetime experiment hence more experimental replicationis needed Also there is a need for testing bacterial growthin dilution cultures nutrients amendment effects of light onrespiration and the size and quality spectra of DOM derivedfrom macrophytes and phytoplankton to better address thissubject Another more speculative hypothesis is a possibleallelopathic effect of macrophytes (mainly submersed) sincethis effect on cyanobacteria is largely known [50 51]This hasbeen considered as a possible explanation for lower bacterialdiversity [52] and metabolism [17] in lake zones extensivelycolonized by macrophytes Direct effects on heterotrophicbacteria however still need to be properly addressed
5 Conclusions
In this study we reported a short-term unchange in macro-phyte-derived DOC availability with exposition to sunlightThis suggests that bacterial and photo-degradation can bevery low under ldquoin siturdquo conditions which presents lowerlight intensity when compared to laboratory experimentsbut more evidence is needed to determine if this is apermanent condition of the system This study adds animportant mechanistic explanation (low reactivity) for theprevious finding that bacterial respiration is lower in littoralwhen compared to pelagic zones in lake Mangueira [53] Wehypothesize that given the climatic conditions continuousgrowth of macrophytes and loading of dissolved recalcitrantcompounds contribute to very slow rates ofDOCdegradationand in a short term slight influence of photo-degradationSubmersed and emergent macrophytes cover extensive areasinside and around the drainage channel where we obtainedthe water for the present experiment and similar extensivemacrophyte coverage is common in many lakes across theworld The acknowledgment and further investigation onthis subject are essential for the understanding of carboncycling and metabolism in systems that are densely coveredby macrophytes
8 Journal of Ecosystems
Conflict of Interests
The authors state no conflict of interests involving any step ofthis study
Acknowledgments
The authors draw particular attention to the assistance pro-vided by theChicoMendes Institute of Biodiversity (ICMBio-ESEC TAIM) Also they sincerely thank Dr Laura Utz forhelping with ciliate sampling and handling and Dr LuizKucharski for providing the scintillation counter They thankthe Conselho Nacional de Desenvolvimento Cientıfico eTecnologico of Brazil (CNPq-Long TermEcological ResearchProgram Site 7 Sistema Hidrologico do Taim) for financialsupport They are also grateful to Dr Janet W Reid (JWRAssociates) for revising the English text
References
[1] R G Wetzel ldquoGradient-dominated ecosystems sources andregulatory functions of dissolved organic matter in freshwaterecosystemsrdquo Hydrobiologia vol 229 no 1 pp 181ndash198 1992
[2] G H Lauster P C Hanson and T K Kratz ldquoGross primaryproduction and respiration differences among littoral andpelagic habitats in northernWisconsin lakesrdquoCanadian Journalof Fisheries and Aquatic Sciences vol 63 no 5 pp 1130ndash11412006
[3] K M Docherty K C Young P A Maurice and S DBridgham ldquoDissolved organicmatter concentration and qualityinfluences upon structure and function of freshwater microbialcommunitiesrdquo Microbial Ecology vol 52 no 3 pp 378ndash3882006
[4] R M W Amon and R Benner ldquoBacterial utilization ofdifferent size classes of dissolved organicmatterrdquo Limnology andOceanography vol 41 no 1 pp 41ndash51 1996
[5] L Bracchini A Cozar AM Dattilo et al ldquoThe role of wetlandsin the chromophoric dissolved organic matter release and itsrelation to aquatic ecosystems optical properties A case ofstudy katonga and Bunjako Bays (Victoria Lake Uganda)rdquoChemosphere vol 63 no 7 pp 1170ndash1178 2006
[6] U Munster and R J Chrost ldquoOrigin composition and micro-bial utilization of dissolved organicmatterrdquo inAquaticMicrobialEcology J Overbeck and R J Chrost Eds pp 8ndash46 SpringerNew York NY USA 1990
[7] D O Hessen ldquoDissolved organic carbon in a humic lake effectson bacterial production and respirationrdquo Hydrobiologia vol229 no 1 pp 115ndash123 1992
[8] M J Lindell W Graneli and L J Tranvik ldquoEnhanced bac-terial growth in response to photochemical transformation ofdissolved organicmatterrdquo Limnology andOceanography vol 40no 1 pp 195ndash199 1995
[9] M A Moran and R G Zepp ldquoRole of photoreactions inthe formation of biologically labile compounds from dissolvedorganicmatterrdquo Limnology and Oceanography vol 42 no 6 pp1307ndash1316 1997
[10] S Bertilsson and L J Tranvik ldquoPhotochemically producedcarboxylic acids as substrates for freshwater bacterioplanktonrdquoLimnology and Oceanography vol 43 no 5 pp 885ndash895 1998
[11] V F Farjalla A M Anesio S Bertilsson and W GranelildquoPhotochemical reactivity of aquatic macrophyte leachates abi-otic transformations and bacterial responserdquo Aquatic MicrobialEcology vol 24 no 2 pp 187ndash195 2001
[12] A P Perez M M Diaz M A Ferraro G C Cusminsky andH E Zagarese ldquoReplicated mesocosm study on the role ofnatural ultraviolet radiation in high CDOM shallow lakesrdquoPhotochemical and Photobiological Sciences vol 2 no 2 pp 118ndash123 2003
[13] A M Amado V F Farjalla F D A Esteves R L BozelliF Roland and A Enrich-Prast ldquoComplementary pathwaysof dissolved organic carbon removal pathways in clear-waterAmazonian ecosystems photochemical degradation and bacte-rial uptakerdquo FEMSMicrobiology Ecology vol 56 no 1 pp 8ndash172006
[14] S G Ribblett M A Palmer and D W Coats ldquoThe importanceof bacterivorous protists in the decomposition of stream leaflitterrdquo Freshwater Biology vol 50 no 3 pp 516ndash526 2005
[15] R A Snyder and M P Hoch ldquoConsequences of protist-stimulated bacterial production for estimating protist growthefficienciesrdquo Hydrobiologia vol 341 no 2 pp 113ndash123 1996
[16] N Rooney and J Kalff ldquoInteractions among epilimneticphosphorus phytoplankton biomass and bacterioplanktonmetabolism in lakes of varying submerged macrophyte coverrdquoHydrobiologia vol 501 pp 75ndash81 2003
[17] H-T Ng D da Motta Marques E Jeppesen and MSoslashndergaard ldquoBacterioplankton in the littoral and pelagic zonesof subtropical shallow lakesrdquo Hydrobiologia vol 646 no 1 pp311ndash326 2010
[18] L O Crossetti L S Cardoso V LM Callegaro et al ldquoInfluenceof the hydrological changes on the phytoplankton structureand dynamics in a subtropical wetland-lake systemrdquo ActaLimnologica Brasiliensia vol 19 pp 315ndash329 2007
[19] C R Fragoso Jr D M L M Marques W Collischonn C EM Tucci and E H vanNes ldquoModelling spatial heterogeneity ofphytoplankton in Lake Mangueira a large shallow subtropicallake in South Brazilrdquo Ecological Modelling vol 219 no 1-2 pp125ndash137 2008
[20] J C Ceballos and M L Rodrigues ldquoEstimativa de insolacaomediante satelite geoestacionario resultados preliminaresrdquo inProceedings of 15th Congresso Brasileiro de Meteorologia INPESao Paulo Brazil 2008
[21] R G Wetzel and G E Likens Limnological Analyses SpringerNew York NY USA 3rd edition 2000
[22] American Public Health Association American Water WorksAssociation and Water Environment Federation StandardMethods For the Examination of Water and Wastewater Amer-ican Public Health Associatio Washington DC USA 20thedition 1999
[23] F J H Mackereth J Heron and J F Talling Water AnalysisSome Revised Methods For Limnologists Freshwater BiologicalAssociation Ambleside UK 2nd edition 1989
[24] D J Strome and M C Miller ldquoPhotolytic changes in dissolvedhumic substancesrdquo Verhandlungen der Internationalen Vereini-gung fur Theoretische und Angewandte Limnologie vol 20 pp1248ndash1254 1978
[25] J R Helms A Stubbins J D Ritchie E C Minor D J Kieberand K Mopper ldquoAbsorption spectral slopes and slope ratiosas indicators of molecular weight source and photobleachingof chromophoric dissolved organic matterrdquo Limnology andOceanography vol 53 no 3 pp 955ndash969 2008
Journal of Ecosystems 9
[26] P AasMM Lyons R Pledger D LMitchell andWH JeffreyldquoInhibition of bacterial activities by solar radiation in nearshorewaters and the Gulf of Mexicordquo Aquatic Microbial Ecology vol11 no 3 pp 229ndash238 1996
[27] R Massana J M Gasol P K Bjoslashrnsen et al ldquoMeasurement ofbacterial size via image analysis of epifluorescence preparationsdescription of an inexpensive system and solutions to some ofthe most common problemsrdquo Scientia Marina vol 61 no 3 pp397ndash407 1997
[28] R L J R Kepner Jr and J R Pratt ldquoUse of fluorochromes fordirect enumeration of total bacteria in environmental samplespast and presentrdquo Microbiological Reviews vol 58 no 4 pp603ndash615 1994
[29] J Liu F B Dazzo O Glagoleva B Yu andA K Jain ldquoCMEIASa computer-aided system for the image analysis of bacterialmorphotypes in microbial communitiesrdquo Microbial Ecologyvol 41 no 3 pp 173ndash194 2001
[30] S Norland ldquoThe relationship between biomass and volume ofbacteriardquo inHandbook of Methods in Aquatic Microbial EcologyP F Kemp B F Sherr E B Sherr and J J Cole Eds pp 339ndash345 Lewis Chelsea Mich USA 1993
[31] D C Simon and F Azam ldquoA simple economical method formeasuring bacterial protein synthesis rates in sea water using3H-leucinerdquo Marine Microbial Food Webs vol 6 pp 107ndash1091992
[32] D Kirchman ldquoMeasuring bacterial biomass production andgrowth rates from leucine incorporation in natural aquatic envi-ronmentsrdquo in MarIne MicrobiologymdashMethods In MicrobiologyJ H Paul Ed pp 227ndash237 Academic Press San Diego CalifUSA 30th edition 2001
[33] H L Golterman R S Clymo and M A M OhnstadMethodsfor Physical and Chemical Analysis of Fresh Waters BlackwellPublishing Company London UK 2nd edition 1978
[34] P Del Giorgio ldquoRapid and precise determination of dissolvedoxygen by spectrophotometry evaluation of interference fromcolor and turbidityrdquo Limnology and Oceanography vol 44 no4 pp 1148ndash1154 1999
[35] P A del Giorgio J J Cole and A Cimbleris ldquoRespiration ratesin bacteria exceed phytoplankton production in unproductiveaquatic systemsrdquo Nature vol 385 no 6612 pp 148ndash151 1997
[36] P A del Giorgio and J J Cole ldquoBacterial growth efficiencyin natural aquatic systemsrdquo Annual Review of Ecology andSystematics vol 29 pp 503ndash541 1998
[37] RDevelopmentCoreTeamRALanguage andEnvironment ForStatistical Computing R Foundation for Statistical ComputingVienna Austria 2011 httpwwwR-projectorg
[38] H J de Lange D P Morris and C E Williamson ldquoSolarultraviolet photodegradation of DOCmay stimulate freshwaterfood websrdquo Journal of Plankton Research vol 25 no 1 pp 111ndash117 2003
[39] K Kalinowska ldquoBacteria nanoflagellates and ciliates as com-ponents of the microbial loop in three lakes of different trophicstatusrdquo Polish Journal of Ecology vol 52 no 1 pp 19ndash34 2004
[40] M A Gates ldquoQuantitative importance of ciliates in the plank-tonic biomass of lake ecosystemsrdquoHydrobiologia vol 108 no 3pp 233ndash238 1984
[41] M K Hamdy and O R Noyes ldquoFormation of methyl mercuryby bacteriardquo Journal of Applied Microbiology vol 30 no 3 pp424ndash432 1975
[42] MRavichandran ldquoInteractions betweenmercury and dissolvedorganic mattermdasha reviewrdquo Chemosphere vol 55 no 3 pp 319ndash331 2004
[43] G M Ferrari ldquoInfluence of pH and heavy metals in thedetermination of yellow substance in estuarine areasrdquo RemoteSensing of Environment vol 37 no 2 pp 89ndash100 1991
[44] A M Anesio J Theil-Nielsen and W Graneli ldquoBacterialgrowth on photochemically transformed leachates from aquaticand terrestrial primary producersrdquo Microbial Ecology vol 40no 3 pp 200ndash208 2000
[45] A M Anesio W Graneli G R Aiken D J Kieber andK Mopper ldquoEffect of humic substance photodegradation onbacterial growth and respiration in lake waterrdquo Applied andEnvironmentalMicrobiology vol 71 no 10 pp 6267ndash6275 2005
[46] S P Seitzinger H Hartnett R Lauck et al ldquoMolecular-level chemical characterization and bioavailability of dissolvedorganic matter in stream water using electrospray-ionizationmass spectrometryrdquo Limnology and Oceanography vol 50 no1 pp 1ndash12 2005
[47] J D Wehr J Petersen and S Findlay ldquoInfluence of threecontrasting detrital carbon sources on planktonic bacterialmetabolism in a mesotrophic lakerdquo Microbial Ecology vol 37no 1 pp 23ndash35 1999
[48] O Holm-Hansen E W Helbling B B Prezelin and RC Smith ldquoPolyethylene bags and solar ultraviolet radiationrdquoScience vol 259 no 5094 pp 534ndash535 1993
[49] K A Kuehn M J Lemke K Suberkropp and R G WetzelldquoMicrobial biomass and production associated with decayingleaf litter of the emergent macrophyte Juncus effususrdquo Limnol-ogy and Oceanography vol 45 no 4 pp 862ndash870 2000
[50] E M Gross S Hilt P Lombardo and G Mulderij ldquoSearch-ing for allelopathic effects of submerged macrophytes onphytoplanktonmdashstate of the art and open questionsrdquo Hydrobi-ologia vol 584 no 1 pp 77ndash88 2007
[51] G Mulderij E H van Nes and E van Donk ldquoMacrophyte-phytoplankton interactions the relative importance of allelopa-thy versus other factorsrdquo Ecological Modelling vol 204 no 1-2pp 85ndash92 2007
[52] Q L Wu G Zwart J Wu M P Kamst-van Agterveld S Liuand M W Hahn ldquoSubmersed macrophytes play a key role instructuring bacterioplankton community composition in thelarge shallow subtropical Taihu Lake Chinardquo EnvironmentalMicrobiology vol 9 no 11 pp 2765ndash2774 2007
[53] N HThey D M Marques and R S Souza ldquoLower respirationin the littoral zone of a subtropical shallow lakerdquo Frontiers inMicrobiology vol 3 p 434 2012
bacterial growth because it releases low-molecular-weightcompounds making them directly available to bacteria [8ndash12] These two routes bacterial and photo-degradationcan also be complementary that is bacteria and light actin concert decomposing different fractions of the DOCpool (autochthonous low-molecular-weight DOC mainlyby bacteria and allochthonous high-molecular-weight DOCmainly by light) [13] Additionally bacterivores (flagellatesand ciliates) can enhance decomposition rates [14] becauseof enhanced bacterial production on protist excretions [15]
Evidence is accumulating that bacterial metabolism canbe lower in the presence of macrophytes at some instancesRooney and Kalff [16] surveyed nine lakes with differentpercentages of macrophyte coverage and found a significantdecrease in the bacterial respiration rate with increasingmacrophyte coverage In southern Brazil a study in shallowcoastal lakes found that bacterial metabolism and biovolumewere frequently lower in littoral than pelagic zones and asso-ciated this with the influence of macrophytes [17] Thereforethe environmental metabolic pathways of the organic matterthat originated from macrophytes are still not understood inthese ecosystems
Previous studies [17] have pointed that the bacterialdegradation of macrophyte-derived carbon is lower in thelittoral zone but a mechanistic explanation is still lackingWe hypothesize that since macrophytes contribute with highloadings of refractory material [5] the photo-degradationmay play a major role in breaking down DOC in littoralzones In the present study we designed three independentexperiments carried out simultaneously to test the role ofbacteria bacteria under grazing pressure and light in thedegradation of DOC derived from macrophytes entering thelittoral zone of the lakeMangueira a large subtropical shallowlake
2 Materials and Methods
21 Study Site Lake Mangueira (80 800 ha state of RioGrande do Sul Southern Brazil) is a large shallow coastalsubtropical lake (Figure 1) Extensive belts of wetlands arelocated north and south of the lake The experiments werecarried out in situ with water from a channel that drainsa large wetland belt into the southernmost part of the lake(coordinates minus33∘311015840454810158401015840 minus53∘71015840566410158401015840) This channel hashigh coverage of submersed macrophytes and the waterentering the lake is highly colored compared to the adjacentwaters in the pelagic zone Hence we assume that mostof the DOC found in this water is mainly originated bymacrophytesThis lake has been investigated for phytoplank-ton zooplankton benthos and ecological modelling [18 19]within theBrazilian Long-TermEcological ResearchProgram(PELD-Taim) and part of the lake lies within a conservationunit (ESEC-Taim) Only one study concerning heterotrophicbacteria in Lake Mangueira has been published so far [17]
The National Institute of Meteorology (INMET) stationlocated at Santa Vitoria do Palmar (minus33∘3110158400010158401015840 minus53∘21101584000Altitude 2401m) provided meteorological data such asmean air temperature (AT) No noticeable precipitation was
recorded during the experiment Global Radiation (GR)(the sum of the radiation that come directly from the sunplus diffused radiation) was estimated by satellite [20] sup-ported by the Brazilian National Institute of Space Research(CPTEC-INPE) Total radiation (accumulated global radia-tion) during the experiment was calculated from the dailymean flux radiation times the hours of sunshine (providedby INMET) Accumulated UV radiation was estimated bymultiplying the total radiation by 23 times 10minus3 which is theproportion of UV radiation in total radiation The dailyUV-index (1 unit = 25mWmminus2) during the experiment wasprovided by INPE (National Institute of Space Researchlthttpwwwinpebrgt)
22 Channel Water Characterization All the following vari-ables were measured in triplicates in the channel waterto characterize the experimental conditions alkalinity wasmeasured through Gran titration method and the pH witha potentiometer (TecnoponMPA 210p Piracicaba SP Brazil)[21] Total solids (TS) weremeasured gravimetrically throughwater evaporation in porcelain dishes [22] Total nitrogen(TN) and total phosphorus (TP) weremeasured by colorime-try [23] Dissolved organic carbon (DOC) was determined bya TOC Analyzer (Shimadzu VCPH Columbia MD USA)as the fraction that passed through a 450∘C precombustedglass fiber filter (Macherey-Nagel GF6 06 120583mmean particleretention size) Absorbance at 430 nm was measured as anestimate of water color and the proportion of low- to high-molecular-weight substances as the ratio Abs250 365 [824] To determine the total density of ciliates 50mL of thesample was fixed with lugol (91 volvol) and samples werestored in the dark and cold Total samples were counted insedimentation chambers under an inverted microscope andreported as individuals times104mminus3 [21] Bacterial productionrespiration and BGE were measured the same way as for theexperiment (see below)
Alkalinity pH Abs430 and Abs250365 were measuredimmediately in the field laboratory Samples for TS TN andTPwere immediately frozen in 1 L polyethylene bottles DOCsampleswere collected in 30mLprecombusted (450∘C for 1 h)amber glass bottles and acidified with H
3PO4
minus3
23 Experimental Procedure Surface water was collected inthe channel with the help of a plastic bucket and broughtto the field laboratory We set up three independent andsimultaneous experiments we separated the water into bulkbacterial and control fractions and measured variables intriplicates in the initial condition and exposed (light) ornot (dark) to sunlight totaling 27 incubation bags Thebulk fraction with no filtration was used to evaluate theeffect of grazing the bacterial fraction was obtained afterfiltration on MN 640d Macherey-Nagel paper filters (meanretention size of 20 to 40120583m) in order to exclude mostbacterivores This unusually permissive retention size wasthe best found to isolate bacteria and retain all metazoansciliates and most flagellates in many previous pilots withdifferent filters (including less permissive ones) ([17] and thisstudy) The control was obtained after the same filtration as
Journal of Ecosystems 3
minus100 minus80 minus60 minus40
minus50
minus40
minus30
minus20
minus10
0
10
Brazil
minus326
minus330
minus334
minus530minus535 minus525
Figure 1 Sampling location the large subtropical shallow LakeMangueira in southern BrazilThe experiment was carried out in its southernpart
the bacterial fraction and addition of the inhibitor HgCl2
(04 120583gmLminus1 final concentration) in order to isolate thephoto-degradation effectThis inhibitor has no effect on pho-toreactivity (the capacity of undergoing oxidation by light)of DOM or UV-visible absorption spectra [25] PolyethyleneWhirl-Pak bags (Nasco Pittsburgh USA) of 500mL capacity(115times23 cm) were filled with 350mL of each fractionThesebags are UV-transparent and did not present differencesin bacterial activity in comparison with quartz containersduring sunlight exposition [26] Apart from the initial bags(9 bags) which were sampled just before the beginning of theincubation 9 bags were protected from sunlight by double-wrapping with aluminum foil the 9 remaining bags wereexposed to natural sunlight Because of the headspace in thebags they floated at the surface and there was no water layerat the upper side of the bags hence we assumed a negligibleabsorbance of UV by the water The bags were randomlydeployed on a plastic tray (1m2times 10 cm high open on theupper side) made of coarse-mesh shade cloth (3 cm times 1 cm)tied to a floating PVC pipe square also 1m2 The tray wasanchored in an unvegetated littoral zone at another pointin the lake (asymp05m depth) away from the sampling pointfor 120 h Farjalla et al [11] conducted an experiment withmacrophyte leachates and bacterial decomposition and foundan endpoint of 96 h of incubation when bacterial numbersreached a plateau related to the total amount of availablegrowth substrate In our study we extended this period byone day because of lack of prior knowledge of what endpointwould apply to our case
24 Experimental Variables The collection and preservationof samplings were carried according to Haig-They [17]Samples for cell counting biovolume and biomass were fixed
in 4 formaldehyde (vol vol) in polyethylene bottles in thefield and stored in the dark at 4∘C until analysis Samplesof the bulk fraction were prefiltered on quantitative paperMN 640d Macherey-Nagel mean mesh size 20 to 40 120583min order to exclude organisms other than bacteria In thelaboratory 2mL of each sample was filtered (lt50 kPa) ina Vacuum Manifold Filtration Tower (Millipore BillericaMA) with 1 mL of Milli-Q water (02120583m filtered) to improvecell dispersion Cells were concentrated on 02 120583m blackpolycarbonate membranes (GE) Approximately 1mL of 10(weight vol) acridine orange stain was added to the filters for5min They were then washed with Milli-Q water and air-dried Filters weremounted on slides withmineral oil (Nujol)and photographed within 3 days A total of 10 images werecaptured per filter and image processing was undertakenon six of them with the help of an image grab systemWe employed a MOTIC 5000 cooled camera coupled to anOlympus IX70 inverted epifluorescence microscope (CenterValley PA USA) Image capture (MOTIC Image 32) andprocessing followed Massana et al [27] with the help of theFreeware Image Tool (v300) The processed and binarizedimages were then used for cell counts and determinationof dimensions Cell density was determined from the meannumber of cells in 6 images and the cell-density equationof Kepner and Pratt [28] Bacterial biovolume (120583mminus3) wasassigned to each cell according to the morphotype [27]utilizing the cell dimensions and morphotypes classificationprovided by CMEIASImage Tool (127) [29] The meanbiovolume per cell was calculated as the mean of all cellsin all images Bacterial biomass (pg C cellminus1) was calculatedemploying an allometric function of biovolume [30] and themean from all cells in all images was multiplied by the celldensity to yield the bacterial carbon concentration of the
4 Journal of Ecosystems
sample (ng C mLminus1) Bacterial production rates (BP) wereestimated through the method of (L[45-3H]) radiolabeledleucine microcentrifugation Incubations for bacterial pro-ductionwere carried out in Eppendorf vials (2mL) and lasted30min inside a tray filledwith lakewater at room temperature(15-16∘C) Radioactivity counts were made after addition of1mL of scintillation liquid (Optiphase HiSafe III Wallac)in a Rack Beta Liquid Scintillation Counter (LKB Wallac1209 Massachusetts USA) for 180 s twice The calculationsassumed the isotopic dilution to be equal to 2 the molar ratioof leucine in the protein pool to be 0073 and the ratio ofcarbonprotein contents to be 0086 [31 32]
The bacterial respiration in the fractions (bulk bacterialand control) was estimated as the rate of oxygen consump-tion through the Winkler method [33] with colorimetricreading [34] This method was conducted separately througha parallel incubation of the fractions in glass BOD bottles of100mL capacity Incubation was done in the dark (double-wrapping the bottles with aluminum foil) with a total of 18bottles (9 initial bottles and 9 final bottles three triplicatesof each of the three fractions) and then only comparisonsamong fractions could be made The bottles were tied bytheir necks to another floating PVC pipe square that wasanchored next to the incubating bags For calculations eachfinal value of O
2was subtracted from the mean of the three
initial bottles and divided by the incubation time assumingconstant respiration rate over time The molar conversionfactor between carbon and oxygen was assumed to be equalto 10 [35]
Bacterial GrowthEfficiency (BGE)was computed accord-ing to equation (I) of del Giorgio et al [36] (I) BGE =(BP)(BP + BR) where BP is the bacterial production rateand BR is the bacterial respiration rate Because respirationwas recorded only for the three fractions also BGE couldonly be calculated and compared among fractions For thecalculations in order to match the results from respirationwe employed the mean of the bacterial production ratebetween the initial and final incubations
Ciliates Abs430 and Abs250 365 were measured thesame way as for field samples [8 21 24]
25 Statistical Treatment Differences in the variables (bacte-rial density biovolume biomass production ciliate densityDOC Abs 430 nm and Abs250 365 nm ratio) among initiallight and dark treatments in each fraction were tested withone-way ANOVA using R 2131 [37] Significant differencesrevealed by ANOVA were tested by Tukeyrsquos a posteriori testIncreases or decreases refer to the light and dark treatmentsin relation to the respective initial condition Bacterial respi-ration and BGE were tested for differences among fractionsand field condition through ANOVA and Tukeyrsquos test
3 Results
The main limnological variables for the experimental condi-tions can be found in Table 1The global cumulative radiation(GR) during the experiment was asymp25 455KJmminus2 Total UVradiation received was estimated to be asymp58 kJmminus2
Bacterial density did not differ among treatments in thebulk fraction it increased with light exposure and decreasedin the dark bags in the bacterial fraction In the controlfraction there was an increase in density in the dark treat-ment Bacterial biovolume did not change with treatmentsBacterial biomass also showed no differences in the bulkfraction It decreased in the dark treatment for the bacterialfraction and increased in the dark treatment in the controlfraction (Table 2)
Bacterial production was unaltered in the bulk fractionIn the bacterial fraction it decreased in the light treatmentand increased in the dark In the control fraction it increasedin the dark Bacterial respiration and BGE did not differamong the fractions (Table 2)
Comparing the bacterial production with the field con-dition only the dark control treatment showed a significantdifference (ANOVA F(920) = 2941 119875 ≪ 0001 dark controlgt Field 119875 ≪ 0001) Bacterial respiration did not differ fromthe field condition in any treatment (ANOVA F(38) = 2517119875 = 0132) BGE decreased in the bulk and bacterial fractionincubations (ANOVAF(38) = 492119875 = 00318 FieldgtBulk119875 = 0058 Field gt Bacterial 119875 = 0052)
In the bulk fraction total ciliate density changed only inthe light treatmentThe light treatment at its turn decreasedcompared to the initial treatment The dark incubation didnot differ statistically from the initial treatmentThe filtrationprocedure successfully excluded ciliates as no ciliates werefound in the bacterial and control fractions any ciliate inthe control was likely lysed by the toxic inhibitor (Table 2)No flagellates were found in the filtered samples undermicroscopic examination
DOC and Abs250 365 showed no effect of treatmentfor any fraction Abs430 (water color) was unaltered in thebulk fraction for all treatments only the bacterial fractionshowed significant color fading in the light treatment (26lower)The control light bags did not show any alterationwithtreatment but the color was less intense than in the dark bagsRates of fading in the light treatment were 00006 00014and 00012 dayminus1 absorbance units for the bulk bacterial andcontrol fractions respectively (Table 2)
4 Discussion
The littoral zones of Lake Mangueira (especially in thenorth and south) are extensively colonized by emergent andsubmersed macrophytes and these plants are expected tocontribute large amounts of organic carbon to the systemMuch of this carbon enters the lake in the form of DOMwhich we found to show little reactivity (did not undergodecomposition by bacteria or light) during short-term incu-bation under in situ conditions We were unable to detectchanges in DOC Abs250 365 and bacterial respirationalthough the water color faded in the light bags Theseresults indicated that photodegradation did occur but didnot modify carbon availability to bacteria Exposure tolight however had contrasting effects it increased bacterialdensity in the bacterial fraction suggesting a positive effectof light but decreased bacterial production in this same
Journal of Ecosystems 5
Table 1 Limnological and biological characterization of the water in the initial conditions of the experiment
1Mean air temperature for the 5 days of incubation2Mean daily flux of GR during incubation (n = 5) 1W = 1 J sminus1 For all other variables n = 33Figures in parentheses are plusmnstandard deviations4119885mean mean depth (m) AT air temperature (∘C) TS total solids (mg Lminus1) Alk alkalinity (120583Eq Lminus1) TN total nitrogen (120583g Lminus1) TP total phosphorus(120583g Lminus1) DOC dissolved organic carbon (mg Lminus1) Abs430 absorbance at 430 nm GR global radiation flux (Wmminus2) LMW low-molecular-weight substances(absorbance at 250 nm)HMW high-molecular-weight substances (absorbance at 365 nm) Abs250 365 (ratio of absorbances at 250 and 365 nm) BP bacterialproduction (120583gC L hminus1) BR bacterial respiration (120583gC L hminus1) BGE bacterial growth efficiency
treatmentfraction suggesting photoinhibition We did notexamine the effect of exposure to light on respiration but ourresults showed no difference in respiration from a previousmeasurement in the field (Table 1) The decrease in BGEwith incubation can be attributed to the increase in respi-ration (even though there was no significant differences inrespiration among fractions it impacted the BGE calculationbecause respiration tended to be higher in bacterial andcontrol fractions)
The general lack of changes in practically all variables inthe bulk fraction indicates that there may be an interactionof grazing with radiation The increase of bacterial densitywith light found in the bacterial fraction may have beenimpeded by grazing in the bulk fraction Higher densitiesof bacteria and bacterial grazers have been found in UV-irradiated DOC suggesting that photo-degradation acts as astimulus to microbial food webs [38] In our case howeverthe decrease in ciliate density with light treatment in thebulk fraction indicated a net effect of photoinhibition onciliates If they did control bacterial density it was at acost of a higher grazing rate per cell On the other side ithas been found that high concentrations of refractory DOClike in humic lakes may be associated to lower bacterialnanoflagellates and ciliates numbers and biomass and hencea less efficient functioning of the microbial loop that islower rates of DOC reincorporation to the food web [39]The density of ciliates in the initial condition (presumably afield concentration estimate) and inside the bags was low (2ndash4 orders of magnitude) when compared to values reportedfor lakes with variable trophic status where they range from19 to 100 times 106 cellsmminus3 (see compilation in Gates [40])Thissuggests that there may be a minor role of ciliates on thedecomposition rates of this refractory DOC in Mangueiralakersquos littoral zones
Unexpectedly we found enhancement in bacterial den-sity biomass and production in the dark treatment of thecontrol fraction This enhancement was first attributed tosome kind of contamination but since no ciliates werefound in these bags and contamination in the three bagssimultaneously is unlikely this result was not disregardedAlthough we were not able to explain this unexpected resultdevelopment of organisms capable of metabolizing mercuryis possible Several common bacterial genera (EscherichiaEnterobacter and Bacillus) are able to resist HgCl
2through
mercury methylation [41]This has compromised the evalua-tion of the photo-degradation alone and needs to be criticallyconsidered
An important point of our study is that we were able tofind significant fading of DOM only in the light treatmentof the bacterial fraction which suggests the existence of acomplementary pathway of dissolved organic degradation bybacteria and light [13] Farjalla et al [11] also found fading(in terms of 250 365 and 430 nm absorbance) of DOM butdecreased bacterial density and production of bacteria inUV-exposed macrophyte leachates indicating that fading itselfsays little about the bioavailability of compounds formedTheincrease in bacterial density together with the stability of theDOC and Abs250 365 suggests that the photodegradationof high-molecular-weight compounds may have producedassimilable substances for bacterial growth [9 10] but theywere readily consumed by bacteria Another important ques-tion still concerning mercury inside control bags is thatthis metal shows great affinity with organic matter alteringthe conformation of the latter [42] other bivalent heavymetals like Cu+2 Pb+2 and Cd+2 have been found to changephotochemistry of yellow substances and humic acids [43]and it is possible that mercury has had some unevaluatednegative effect on photochemical reactivity in the bags whereit was added to
Simultaneous irradiation and incubation in our studymay have exposed bacteria to continuous production ofinhibitory free radicals as suggested by the decrease inbacterial production Many studies that dealt with bacterialutilization of photochemically transformed organic carbonderived from macrophytes employed an approach of pre-treating the leachates with UV light prior to the incubationof bacterial batches [11 13 44] In many cases this priorexposure has been found to inhibit bacterial production anddecrease growth efficiency at least for a short period whichhas been attributed to the formation of hydrogen peroxideand possibly other free radicals by light [11 45] Formationof nonassimilable compounds is also possible Seitzingeret al [46] studied bacterial utilization of dissolved organicmatter in two streams and found that BP increased greatlyduring two days of incubation while DOC concentrationsignificantly decreased (45ndash50) in 12 days However 60of the compounds found in the complex mixture of organicmolecules were not consumed which Seitzinger et al [46]
6 Journal of Ecosystems
Table2Differencesb
etweeninitialcond
ition
andexpo
sition(light)or
not(dark)o
fmacroph
yte-deriv
edDOM
tosunlight
inun
filteredwater
(Bulk)filteredwater
(Bacteria
l)andCon
trol
(HgC
l 2)for120
hun
derinsitucond
ition
sinthesouthern
partof
thesubtropicalshallo
wlake
Mangueira
(sou
thernBrazil)
teste
dby
one-way
ANOVA
andTu
keyrsquos
test(m
eanplusmnsta
ndard
deviation)
Experim
ent
(I)B
ulk
(II)Ba
cterial
(III)C
ontro
lInitial
Light
Dark
Initial
Light
Dark
Initial
Light
Dark
Bacteriald
ensity
(cells10
6mLminus
1 )081plusmn016
a076plusmn050
a051plusmn041
a17
1plusmn031
a265plusmn009
b063plusmn007
c14
3plusmn013
a10
3plusmn010
a218plusmn033
b
Bacterialbiovolume
(120583m
3 )006plusmn002
a005plusmn001
a005plusmn001
a010plusmn001
a008plusmn001
a006plusmn001
a006plusmn001
a007plusmn002
a007plusmn003
a
Bacterialbiomass
(ngC
mLminus
1 )65plusmn026
a372plusmn206
a333plusmn320
a1864plusmn591
a2473plusmn277
a414plusmn045
b95
7plusmn13
1a807plusmn18
5a1649plusmn357
b
Bacterialprodu
ction
(120583gC
Lminus1 hminus1 )
012plusmn004
a028plusmn024
a015plusmn003
a015plusmn001
a002plusmn002
b014plusmn002
a003plusmn001
a003plusmn002
a077plusmn020
b
Bacterialrespiratio
n(120583gC
Lminus1 hminus1 )
NA
NA
109plusmn012
aNA
NA
228plusmn088
aNA
NA
214plusmn12
7a
BacterialG
rowth
Efficiency
NA
NA
011plusmn002
aNA
NA
007plusmn003
aNA
NA
022plusmn012
a
Ciliatedensity
(times10
4cells
mminus3 )
414plusmn18
0a18
0plusmn312
b1560plusmn178a
0plusmn0
0plusmn0
0plusmn0
0plusmn0
0plusmn0
0plusmn0
Diss
olvedOrganicCa
rbon
(mgC
Lminus1 )
1869plusmn087
a1946plusmn112
a2626plusmn78
3a2507plusmn136
a4358plusmn255
a3534plusmn204
a2090plusmn354
a3575plusmn367
a46
10plusmn175a
Abs430
(Abs
430n
m(times10minus2))
251plusmn022
a221plusmn012
a263plusmn019
a269plusmn006
a19
8plusmn014
b235plusmn020
a244plusmn016
ab18
4plusmn035
b271plusmn025
a
Abs250
365
nmratio
085plusmn009
a085plusmn002
a085plusmn004
a076plusmn007
a079plusmn018
a087plusmn002
a075plusmn005
a079plusmn025
a087plusmn006
a
Sign
ificanceo
fone-w
ayANOVA
andTu
keyrsquos
aposte
rioritestatPlt005
isindicatedwith
different
lowercase
superscriptletterswith
ineach
experim
ent(Initialversus
LightversusD
ark)Th
eexceptio
nisbacterial
respira
tionandgrow
theffi
ciencyw
here
differences
weretestedam
ongexperim
ents
NAn
otavailableb
ecause
respira
tionwas
measuredin
dark
bottlesFor
allvariables
n=3replicates
Journal of Ecosystems 7
suggested may have resulted from some inhibition factorinvolved in their utilization Contrastingly Perez et al [12]exposed natural river-water DOM to light also in a simulta-neous exposure and found bacterial enhancement with UVexposure These differences could be due to variations inorigin age and biochemical composition of the DOM [45]
The generally low bioavailability of DOC in the formof humic substances [6] indicates that a large proportionof the dissolved carbon in Lake Mangueira is refractory tobacterial consumption This Abs250 365 ratio is higher inthe littoral zone than in the pelagic zone suggesting higherproportion of low-molecular-weight (LMW Abs 250 nm)substances in the littoral zone when compared to the pelagiczone In fact this was confirmed (pelagic Abs250 mean= 003 standard deviation = 0002 F(14) = 1163 119875 lt0001 littoral zone data obtained from Table 1) In spiteof the smaller size of their molecules these substances canbe less reactive Amon and Benner [4] conducted a cross-environment experiment where they analyzed the reactivityof low-(LMW lt1 KDa) and high-molecular-weight (HMWgt1 KDa) dissolved compounds and found significant changesin BP and BR during the experiment (also five days)Unexpectedly these bacterial variables were higher in HMWsubstrates and based on these results the authors proposedthe size-reactivity continuum model that predicts that themajor path of degradation goes from large highly reactiveto small highly recalcitrant molecules If this hypothesis isbroadly applicable to many ecosystems it suggests that inLakeMangueira the compounds lixiviated by themacrophytebelt had already degraded to some extent and accumulatedas dissolved unreactive LMW compounds with very lowdegradation rates in the littoral zones
The age of the DOM subjected to bacterial degradationcan be fundamental for its availability In our experiment wefocused on the subsequent stage of macrophyte degradationthat is after bacterial and light reworking had alreadyoccurred inside the macrophyte stands Most studies thatinvestigated microbial degradation of macrophytes focusedon the early stages of degradation with exposure of coarsefragments of macrophytes to leaching For example Wehret al [47] found a 45-fold increase in bacterial productionafter 168 h of incubation of macrophyte detritus ConverselyHolm-Hansen et al [48] demonstrated very low rates ofmicrobial degradation of the emergent macrophyte Juncuseffusus only 23of the total biomass in leaf litter bagswas lostafter 268 days Enhancement of bacterial production mightbe related to the early stage of degradation given that Kuehnet al [49] employed senescent leaves in their experiment asituation more similar to ours
One possible explanation for the low photodegradationrates may be the total amount of UV accumulated during theexperiment In the study of Farjalla et al [11] macrophyteleachates were photochemically transformed with total UVenergy asymp5670KJmminus2 which was able to induce a bacterialpositive response This UV intensity is almost 100 timeshigher than that estimated for the field conditions in thepresent study (asymp58KJmminus2) One implication is that underlaboratory conditions the rates of photo-degradation may
be overestimated compared to natural conditions Anotherimportant point however is that polyethylene bags used inthis study may present a reduced rate of UV transmittance(eg 68 of UVB at 300 nm) [48] Even though the trans-mittance increases slightly towards larger wavelengths [26]this quenching may have imposed slightly reduced rates ofphoto-degradation than in the field
We attempted to determine the importance of the twomain routes of carbon degradation in aquatic systemsthat is bacterial and photo-degradation on macrophyte-derived DOM flowing into Lake Mangueira from a largewetland belt Remarkably our results showed few changesin environmental parameters related to carbon degradation(DOC Abs250 365 and respiration) even though bacterialvariables did show changes under lightdark incubationconditions (120 h) This DOM that enters the lake seemsto be highly unreactive in a short-term and may be thecause for the difference in bacterial metabolism between thelittoral and pelagic zones reported by Ng et al [17] sincethis is the main carbon source for bacteria in the littoralzone It is important to take into consideration that it wasthe first approach to the subject in this lake and was aonetime experiment hence more experimental replicationis needed Also there is a need for testing bacterial growthin dilution cultures nutrients amendment effects of light onrespiration and the size and quality spectra of DOM derivedfrom macrophytes and phytoplankton to better address thissubject Another more speculative hypothesis is a possibleallelopathic effect of macrophytes (mainly submersed) sincethis effect on cyanobacteria is largely known [50 51]This hasbeen considered as a possible explanation for lower bacterialdiversity [52] and metabolism [17] in lake zones extensivelycolonized by macrophytes Direct effects on heterotrophicbacteria however still need to be properly addressed
5 Conclusions
In this study we reported a short-term unchange in macro-phyte-derived DOC availability with exposition to sunlightThis suggests that bacterial and photo-degradation can bevery low under ldquoin siturdquo conditions which presents lowerlight intensity when compared to laboratory experimentsbut more evidence is needed to determine if this is apermanent condition of the system This study adds animportant mechanistic explanation (low reactivity) for theprevious finding that bacterial respiration is lower in littoralwhen compared to pelagic zones in lake Mangueira [53] Wehypothesize that given the climatic conditions continuousgrowth of macrophytes and loading of dissolved recalcitrantcompounds contribute to very slow rates ofDOCdegradationand in a short term slight influence of photo-degradationSubmersed and emergent macrophytes cover extensive areasinside and around the drainage channel where we obtainedthe water for the present experiment and similar extensivemacrophyte coverage is common in many lakes across theworld The acknowledgment and further investigation onthis subject are essential for the understanding of carboncycling and metabolism in systems that are densely coveredby macrophytes
8 Journal of Ecosystems
Conflict of Interests
The authors state no conflict of interests involving any step ofthis study
Acknowledgments
The authors draw particular attention to the assistance pro-vided by theChicoMendes Institute of Biodiversity (ICMBio-ESEC TAIM) Also they sincerely thank Dr Laura Utz forhelping with ciliate sampling and handling and Dr LuizKucharski for providing the scintillation counter They thankthe Conselho Nacional de Desenvolvimento Cientıfico eTecnologico of Brazil (CNPq-Long TermEcological ResearchProgram Site 7 Sistema Hidrologico do Taim) for financialsupport They are also grateful to Dr Janet W Reid (JWRAssociates) for revising the English text
References
[1] R G Wetzel ldquoGradient-dominated ecosystems sources andregulatory functions of dissolved organic matter in freshwaterecosystemsrdquo Hydrobiologia vol 229 no 1 pp 181ndash198 1992
[2] G H Lauster P C Hanson and T K Kratz ldquoGross primaryproduction and respiration differences among littoral andpelagic habitats in northernWisconsin lakesrdquoCanadian Journalof Fisheries and Aquatic Sciences vol 63 no 5 pp 1130ndash11412006
[3] K M Docherty K C Young P A Maurice and S DBridgham ldquoDissolved organicmatter concentration and qualityinfluences upon structure and function of freshwater microbialcommunitiesrdquo Microbial Ecology vol 52 no 3 pp 378ndash3882006
[4] R M W Amon and R Benner ldquoBacterial utilization ofdifferent size classes of dissolved organicmatterrdquo Limnology andOceanography vol 41 no 1 pp 41ndash51 1996
[5] L Bracchini A Cozar AM Dattilo et al ldquoThe role of wetlandsin the chromophoric dissolved organic matter release and itsrelation to aquatic ecosystems optical properties A case ofstudy katonga and Bunjako Bays (Victoria Lake Uganda)rdquoChemosphere vol 63 no 7 pp 1170ndash1178 2006
[6] U Munster and R J Chrost ldquoOrigin composition and micro-bial utilization of dissolved organicmatterrdquo inAquaticMicrobialEcology J Overbeck and R J Chrost Eds pp 8ndash46 SpringerNew York NY USA 1990
[7] D O Hessen ldquoDissolved organic carbon in a humic lake effectson bacterial production and respirationrdquo Hydrobiologia vol229 no 1 pp 115ndash123 1992
[8] M J Lindell W Graneli and L J Tranvik ldquoEnhanced bac-terial growth in response to photochemical transformation ofdissolved organicmatterrdquo Limnology andOceanography vol 40no 1 pp 195ndash199 1995
[9] M A Moran and R G Zepp ldquoRole of photoreactions inthe formation of biologically labile compounds from dissolvedorganicmatterrdquo Limnology and Oceanography vol 42 no 6 pp1307ndash1316 1997
[10] S Bertilsson and L J Tranvik ldquoPhotochemically producedcarboxylic acids as substrates for freshwater bacterioplanktonrdquoLimnology and Oceanography vol 43 no 5 pp 885ndash895 1998
[11] V F Farjalla A M Anesio S Bertilsson and W GranelildquoPhotochemical reactivity of aquatic macrophyte leachates abi-otic transformations and bacterial responserdquo Aquatic MicrobialEcology vol 24 no 2 pp 187ndash195 2001
[12] A P Perez M M Diaz M A Ferraro G C Cusminsky andH E Zagarese ldquoReplicated mesocosm study on the role ofnatural ultraviolet radiation in high CDOM shallow lakesrdquoPhotochemical and Photobiological Sciences vol 2 no 2 pp 118ndash123 2003
[13] A M Amado V F Farjalla F D A Esteves R L BozelliF Roland and A Enrich-Prast ldquoComplementary pathwaysof dissolved organic carbon removal pathways in clear-waterAmazonian ecosystems photochemical degradation and bacte-rial uptakerdquo FEMSMicrobiology Ecology vol 56 no 1 pp 8ndash172006
[14] S G Ribblett M A Palmer and D W Coats ldquoThe importanceof bacterivorous protists in the decomposition of stream leaflitterrdquo Freshwater Biology vol 50 no 3 pp 516ndash526 2005
[15] R A Snyder and M P Hoch ldquoConsequences of protist-stimulated bacterial production for estimating protist growthefficienciesrdquo Hydrobiologia vol 341 no 2 pp 113ndash123 1996
[16] N Rooney and J Kalff ldquoInteractions among epilimneticphosphorus phytoplankton biomass and bacterioplanktonmetabolism in lakes of varying submerged macrophyte coverrdquoHydrobiologia vol 501 pp 75ndash81 2003
[17] H-T Ng D da Motta Marques E Jeppesen and MSoslashndergaard ldquoBacterioplankton in the littoral and pelagic zonesof subtropical shallow lakesrdquo Hydrobiologia vol 646 no 1 pp311ndash326 2010
[18] L O Crossetti L S Cardoso V LM Callegaro et al ldquoInfluenceof the hydrological changes on the phytoplankton structureand dynamics in a subtropical wetland-lake systemrdquo ActaLimnologica Brasiliensia vol 19 pp 315ndash329 2007
[19] C R Fragoso Jr D M L M Marques W Collischonn C EM Tucci and E H vanNes ldquoModelling spatial heterogeneity ofphytoplankton in Lake Mangueira a large shallow subtropicallake in South Brazilrdquo Ecological Modelling vol 219 no 1-2 pp125ndash137 2008
[20] J C Ceballos and M L Rodrigues ldquoEstimativa de insolacaomediante satelite geoestacionario resultados preliminaresrdquo inProceedings of 15th Congresso Brasileiro de Meteorologia INPESao Paulo Brazil 2008
[21] R G Wetzel and G E Likens Limnological Analyses SpringerNew York NY USA 3rd edition 2000
[22] American Public Health Association American Water WorksAssociation and Water Environment Federation StandardMethods For the Examination of Water and Wastewater Amer-ican Public Health Associatio Washington DC USA 20thedition 1999
[23] F J H Mackereth J Heron and J F Talling Water AnalysisSome Revised Methods For Limnologists Freshwater BiologicalAssociation Ambleside UK 2nd edition 1989
[24] D J Strome and M C Miller ldquoPhotolytic changes in dissolvedhumic substancesrdquo Verhandlungen der Internationalen Vereini-gung fur Theoretische und Angewandte Limnologie vol 20 pp1248ndash1254 1978
[25] J R Helms A Stubbins J D Ritchie E C Minor D J Kieberand K Mopper ldquoAbsorption spectral slopes and slope ratiosas indicators of molecular weight source and photobleachingof chromophoric dissolved organic matterrdquo Limnology andOceanography vol 53 no 3 pp 955ndash969 2008
Journal of Ecosystems 9
[26] P AasMM Lyons R Pledger D LMitchell andWH JeffreyldquoInhibition of bacterial activities by solar radiation in nearshorewaters and the Gulf of Mexicordquo Aquatic Microbial Ecology vol11 no 3 pp 229ndash238 1996
[27] R Massana J M Gasol P K Bjoslashrnsen et al ldquoMeasurement ofbacterial size via image analysis of epifluorescence preparationsdescription of an inexpensive system and solutions to some ofthe most common problemsrdquo Scientia Marina vol 61 no 3 pp397ndash407 1997
[28] R L J R Kepner Jr and J R Pratt ldquoUse of fluorochromes fordirect enumeration of total bacteria in environmental samplespast and presentrdquo Microbiological Reviews vol 58 no 4 pp603ndash615 1994
[29] J Liu F B Dazzo O Glagoleva B Yu andA K Jain ldquoCMEIASa computer-aided system for the image analysis of bacterialmorphotypes in microbial communitiesrdquo Microbial Ecologyvol 41 no 3 pp 173ndash194 2001
[30] S Norland ldquoThe relationship between biomass and volume ofbacteriardquo inHandbook of Methods in Aquatic Microbial EcologyP F Kemp B F Sherr E B Sherr and J J Cole Eds pp 339ndash345 Lewis Chelsea Mich USA 1993
[31] D C Simon and F Azam ldquoA simple economical method formeasuring bacterial protein synthesis rates in sea water using3H-leucinerdquo Marine Microbial Food Webs vol 6 pp 107ndash1091992
[32] D Kirchman ldquoMeasuring bacterial biomass production andgrowth rates from leucine incorporation in natural aquatic envi-ronmentsrdquo in MarIne MicrobiologymdashMethods In MicrobiologyJ H Paul Ed pp 227ndash237 Academic Press San Diego CalifUSA 30th edition 2001
[33] H L Golterman R S Clymo and M A M OhnstadMethodsfor Physical and Chemical Analysis of Fresh Waters BlackwellPublishing Company London UK 2nd edition 1978
[34] P Del Giorgio ldquoRapid and precise determination of dissolvedoxygen by spectrophotometry evaluation of interference fromcolor and turbidityrdquo Limnology and Oceanography vol 44 no4 pp 1148ndash1154 1999
[35] P A del Giorgio J J Cole and A Cimbleris ldquoRespiration ratesin bacteria exceed phytoplankton production in unproductiveaquatic systemsrdquo Nature vol 385 no 6612 pp 148ndash151 1997
[36] P A del Giorgio and J J Cole ldquoBacterial growth efficiencyin natural aquatic systemsrdquo Annual Review of Ecology andSystematics vol 29 pp 503ndash541 1998
[37] RDevelopmentCoreTeamRALanguage andEnvironment ForStatistical Computing R Foundation for Statistical ComputingVienna Austria 2011 httpwwwR-projectorg
[38] H J de Lange D P Morris and C E Williamson ldquoSolarultraviolet photodegradation of DOCmay stimulate freshwaterfood websrdquo Journal of Plankton Research vol 25 no 1 pp 111ndash117 2003
[39] K Kalinowska ldquoBacteria nanoflagellates and ciliates as com-ponents of the microbial loop in three lakes of different trophicstatusrdquo Polish Journal of Ecology vol 52 no 1 pp 19ndash34 2004
[40] M A Gates ldquoQuantitative importance of ciliates in the plank-tonic biomass of lake ecosystemsrdquoHydrobiologia vol 108 no 3pp 233ndash238 1984
[41] M K Hamdy and O R Noyes ldquoFormation of methyl mercuryby bacteriardquo Journal of Applied Microbiology vol 30 no 3 pp424ndash432 1975
[42] MRavichandran ldquoInteractions betweenmercury and dissolvedorganic mattermdasha reviewrdquo Chemosphere vol 55 no 3 pp 319ndash331 2004
[43] G M Ferrari ldquoInfluence of pH and heavy metals in thedetermination of yellow substance in estuarine areasrdquo RemoteSensing of Environment vol 37 no 2 pp 89ndash100 1991
[44] A M Anesio J Theil-Nielsen and W Graneli ldquoBacterialgrowth on photochemically transformed leachates from aquaticand terrestrial primary producersrdquo Microbial Ecology vol 40no 3 pp 200ndash208 2000
[45] A M Anesio W Graneli G R Aiken D J Kieber andK Mopper ldquoEffect of humic substance photodegradation onbacterial growth and respiration in lake waterrdquo Applied andEnvironmentalMicrobiology vol 71 no 10 pp 6267ndash6275 2005
[46] S P Seitzinger H Hartnett R Lauck et al ldquoMolecular-level chemical characterization and bioavailability of dissolvedorganic matter in stream water using electrospray-ionizationmass spectrometryrdquo Limnology and Oceanography vol 50 no1 pp 1ndash12 2005
[47] J D Wehr J Petersen and S Findlay ldquoInfluence of threecontrasting detrital carbon sources on planktonic bacterialmetabolism in a mesotrophic lakerdquo Microbial Ecology vol 37no 1 pp 23ndash35 1999
[48] O Holm-Hansen E W Helbling B B Prezelin and RC Smith ldquoPolyethylene bags and solar ultraviolet radiationrdquoScience vol 259 no 5094 pp 534ndash535 1993
[49] K A Kuehn M J Lemke K Suberkropp and R G WetzelldquoMicrobial biomass and production associated with decayingleaf litter of the emergent macrophyte Juncus effususrdquo Limnol-ogy and Oceanography vol 45 no 4 pp 862ndash870 2000
[50] E M Gross S Hilt P Lombardo and G Mulderij ldquoSearch-ing for allelopathic effects of submerged macrophytes onphytoplanktonmdashstate of the art and open questionsrdquo Hydrobi-ologia vol 584 no 1 pp 77ndash88 2007
[51] G Mulderij E H van Nes and E van Donk ldquoMacrophyte-phytoplankton interactions the relative importance of allelopa-thy versus other factorsrdquo Ecological Modelling vol 204 no 1-2pp 85ndash92 2007
[52] Q L Wu G Zwart J Wu M P Kamst-van Agterveld S Liuand M W Hahn ldquoSubmersed macrophytes play a key role instructuring bacterioplankton community composition in thelarge shallow subtropical Taihu Lake Chinardquo EnvironmentalMicrobiology vol 9 no 11 pp 2765ndash2774 2007
[53] N HThey D M Marques and R S Souza ldquoLower respirationin the littoral zone of a subtropical shallow lakerdquo Frontiers inMicrobiology vol 3 p 434 2012
Figure 1 Sampling location the large subtropical shallow LakeMangueira in southern BrazilThe experiment was carried out in its southernpart
the bacterial fraction and addition of the inhibitor HgCl2
(04 120583gmLminus1 final concentration) in order to isolate thephoto-degradation effectThis inhibitor has no effect on pho-toreactivity (the capacity of undergoing oxidation by light)of DOM or UV-visible absorption spectra [25] PolyethyleneWhirl-Pak bags (Nasco Pittsburgh USA) of 500mL capacity(115times23 cm) were filled with 350mL of each fractionThesebags are UV-transparent and did not present differencesin bacterial activity in comparison with quartz containersduring sunlight exposition [26] Apart from the initial bags(9 bags) which were sampled just before the beginning of theincubation 9 bags were protected from sunlight by double-wrapping with aluminum foil the 9 remaining bags wereexposed to natural sunlight Because of the headspace in thebags they floated at the surface and there was no water layerat the upper side of the bags hence we assumed a negligibleabsorbance of UV by the water The bags were randomlydeployed on a plastic tray (1m2times 10 cm high open on theupper side) made of coarse-mesh shade cloth (3 cm times 1 cm)tied to a floating PVC pipe square also 1m2 The tray wasanchored in an unvegetated littoral zone at another pointin the lake (asymp05m depth) away from the sampling pointfor 120 h Farjalla et al [11] conducted an experiment withmacrophyte leachates and bacterial decomposition and foundan endpoint of 96 h of incubation when bacterial numbersreached a plateau related to the total amount of availablegrowth substrate In our study we extended this period byone day because of lack of prior knowledge of what endpointwould apply to our case
24 Experimental Variables The collection and preservationof samplings were carried according to Haig-They [17]Samples for cell counting biovolume and biomass were fixed
in 4 formaldehyde (vol vol) in polyethylene bottles in thefield and stored in the dark at 4∘C until analysis Samplesof the bulk fraction were prefiltered on quantitative paperMN 640d Macherey-Nagel mean mesh size 20 to 40 120583min order to exclude organisms other than bacteria In thelaboratory 2mL of each sample was filtered (lt50 kPa) ina Vacuum Manifold Filtration Tower (Millipore BillericaMA) with 1 mL of Milli-Q water (02120583m filtered) to improvecell dispersion Cells were concentrated on 02 120583m blackpolycarbonate membranes (GE) Approximately 1mL of 10(weight vol) acridine orange stain was added to the filters for5min They were then washed with Milli-Q water and air-dried Filters weremounted on slides withmineral oil (Nujol)and photographed within 3 days A total of 10 images werecaptured per filter and image processing was undertakenon six of them with the help of an image grab systemWe employed a MOTIC 5000 cooled camera coupled to anOlympus IX70 inverted epifluorescence microscope (CenterValley PA USA) Image capture (MOTIC Image 32) andprocessing followed Massana et al [27] with the help of theFreeware Image Tool (v300) The processed and binarizedimages were then used for cell counts and determinationof dimensions Cell density was determined from the meannumber of cells in 6 images and the cell-density equationof Kepner and Pratt [28] Bacterial biovolume (120583mminus3) wasassigned to each cell according to the morphotype [27]utilizing the cell dimensions and morphotypes classificationprovided by CMEIASImage Tool (127) [29] The meanbiovolume per cell was calculated as the mean of all cellsin all images Bacterial biomass (pg C cellminus1) was calculatedemploying an allometric function of biovolume [30] and themean from all cells in all images was multiplied by the celldensity to yield the bacterial carbon concentration of the
4 Journal of Ecosystems
sample (ng C mLminus1) Bacterial production rates (BP) wereestimated through the method of (L[45-3H]) radiolabeledleucine microcentrifugation Incubations for bacterial pro-ductionwere carried out in Eppendorf vials (2mL) and lasted30min inside a tray filledwith lakewater at room temperature(15-16∘C) Radioactivity counts were made after addition of1mL of scintillation liquid (Optiphase HiSafe III Wallac)in a Rack Beta Liquid Scintillation Counter (LKB Wallac1209 Massachusetts USA) for 180 s twice The calculationsassumed the isotopic dilution to be equal to 2 the molar ratioof leucine in the protein pool to be 0073 and the ratio ofcarbonprotein contents to be 0086 [31 32]
The bacterial respiration in the fractions (bulk bacterialand control) was estimated as the rate of oxygen consump-tion through the Winkler method [33] with colorimetricreading [34] This method was conducted separately througha parallel incubation of the fractions in glass BOD bottles of100mL capacity Incubation was done in the dark (double-wrapping the bottles with aluminum foil) with a total of 18bottles (9 initial bottles and 9 final bottles three triplicatesof each of the three fractions) and then only comparisonsamong fractions could be made The bottles were tied bytheir necks to another floating PVC pipe square that wasanchored next to the incubating bags For calculations eachfinal value of O
2was subtracted from the mean of the three
initial bottles and divided by the incubation time assumingconstant respiration rate over time The molar conversionfactor between carbon and oxygen was assumed to be equalto 10 [35]
Bacterial GrowthEfficiency (BGE)was computed accord-ing to equation (I) of del Giorgio et al [36] (I) BGE =(BP)(BP + BR) where BP is the bacterial production rateand BR is the bacterial respiration rate Because respirationwas recorded only for the three fractions also BGE couldonly be calculated and compared among fractions For thecalculations in order to match the results from respirationwe employed the mean of the bacterial production ratebetween the initial and final incubations
Ciliates Abs430 and Abs250 365 were measured thesame way as for field samples [8 21 24]
25 Statistical Treatment Differences in the variables (bacte-rial density biovolume biomass production ciliate densityDOC Abs 430 nm and Abs250 365 nm ratio) among initiallight and dark treatments in each fraction were tested withone-way ANOVA using R 2131 [37] Significant differencesrevealed by ANOVA were tested by Tukeyrsquos a posteriori testIncreases or decreases refer to the light and dark treatmentsin relation to the respective initial condition Bacterial respi-ration and BGE were tested for differences among fractionsand field condition through ANOVA and Tukeyrsquos test
3 Results
The main limnological variables for the experimental condi-tions can be found in Table 1The global cumulative radiation(GR) during the experiment was asymp25 455KJmminus2 Total UVradiation received was estimated to be asymp58 kJmminus2
Bacterial density did not differ among treatments in thebulk fraction it increased with light exposure and decreasedin the dark bags in the bacterial fraction In the controlfraction there was an increase in density in the dark treat-ment Bacterial biovolume did not change with treatmentsBacterial biomass also showed no differences in the bulkfraction It decreased in the dark treatment for the bacterialfraction and increased in the dark treatment in the controlfraction (Table 2)
Bacterial production was unaltered in the bulk fractionIn the bacterial fraction it decreased in the light treatmentand increased in the dark In the control fraction it increasedin the dark Bacterial respiration and BGE did not differamong the fractions (Table 2)
Comparing the bacterial production with the field con-dition only the dark control treatment showed a significantdifference (ANOVA F(920) = 2941 119875 ≪ 0001 dark controlgt Field 119875 ≪ 0001) Bacterial respiration did not differ fromthe field condition in any treatment (ANOVA F(38) = 2517119875 = 0132) BGE decreased in the bulk and bacterial fractionincubations (ANOVAF(38) = 492119875 = 00318 FieldgtBulk119875 = 0058 Field gt Bacterial 119875 = 0052)
In the bulk fraction total ciliate density changed only inthe light treatmentThe light treatment at its turn decreasedcompared to the initial treatment The dark incubation didnot differ statistically from the initial treatmentThe filtrationprocedure successfully excluded ciliates as no ciliates werefound in the bacterial and control fractions any ciliate inthe control was likely lysed by the toxic inhibitor (Table 2)No flagellates were found in the filtered samples undermicroscopic examination
DOC and Abs250 365 showed no effect of treatmentfor any fraction Abs430 (water color) was unaltered in thebulk fraction for all treatments only the bacterial fractionshowed significant color fading in the light treatment (26lower)The control light bags did not show any alterationwithtreatment but the color was less intense than in the dark bagsRates of fading in the light treatment were 00006 00014and 00012 dayminus1 absorbance units for the bulk bacterial andcontrol fractions respectively (Table 2)
4 Discussion
The littoral zones of Lake Mangueira (especially in thenorth and south) are extensively colonized by emergent andsubmersed macrophytes and these plants are expected tocontribute large amounts of organic carbon to the systemMuch of this carbon enters the lake in the form of DOMwhich we found to show little reactivity (did not undergodecomposition by bacteria or light) during short-term incu-bation under in situ conditions We were unable to detectchanges in DOC Abs250 365 and bacterial respirationalthough the water color faded in the light bags Theseresults indicated that photodegradation did occur but didnot modify carbon availability to bacteria Exposure tolight however had contrasting effects it increased bacterialdensity in the bacterial fraction suggesting a positive effectof light but decreased bacterial production in this same
Journal of Ecosystems 5
Table 1 Limnological and biological characterization of the water in the initial conditions of the experiment
1Mean air temperature for the 5 days of incubation2Mean daily flux of GR during incubation (n = 5) 1W = 1 J sminus1 For all other variables n = 33Figures in parentheses are plusmnstandard deviations4119885mean mean depth (m) AT air temperature (∘C) TS total solids (mg Lminus1) Alk alkalinity (120583Eq Lminus1) TN total nitrogen (120583g Lminus1) TP total phosphorus(120583g Lminus1) DOC dissolved organic carbon (mg Lminus1) Abs430 absorbance at 430 nm GR global radiation flux (Wmminus2) LMW low-molecular-weight substances(absorbance at 250 nm)HMW high-molecular-weight substances (absorbance at 365 nm) Abs250 365 (ratio of absorbances at 250 and 365 nm) BP bacterialproduction (120583gC L hminus1) BR bacterial respiration (120583gC L hminus1) BGE bacterial growth efficiency
treatmentfraction suggesting photoinhibition We did notexamine the effect of exposure to light on respiration but ourresults showed no difference in respiration from a previousmeasurement in the field (Table 1) The decrease in BGEwith incubation can be attributed to the increase in respi-ration (even though there was no significant differences inrespiration among fractions it impacted the BGE calculationbecause respiration tended to be higher in bacterial andcontrol fractions)
The general lack of changes in practically all variables inthe bulk fraction indicates that there may be an interactionof grazing with radiation The increase of bacterial densitywith light found in the bacterial fraction may have beenimpeded by grazing in the bulk fraction Higher densitiesof bacteria and bacterial grazers have been found in UV-irradiated DOC suggesting that photo-degradation acts as astimulus to microbial food webs [38] In our case howeverthe decrease in ciliate density with light treatment in thebulk fraction indicated a net effect of photoinhibition onciliates If they did control bacterial density it was at acost of a higher grazing rate per cell On the other side ithas been found that high concentrations of refractory DOClike in humic lakes may be associated to lower bacterialnanoflagellates and ciliates numbers and biomass and hencea less efficient functioning of the microbial loop that islower rates of DOC reincorporation to the food web [39]The density of ciliates in the initial condition (presumably afield concentration estimate) and inside the bags was low (2ndash4 orders of magnitude) when compared to values reportedfor lakes with variable trophic status where they range from19 to 100 times 106 cellsmminus3 (see compilation in Gates [40])Thissuggests that there may be a minor role of ciliates on thedecomposition rates of this refractory DOC in Mangueiralakersquos littoral zones
Unexpectedly we found enhancement in bacterial den-sity biomass and production in the dark treatment of thecontrol fraction This enhancement was first attributed tosome kind of contamination but since no ciliates werefound in these bags and contamination in the three bagssimultaneously is unlikely this result was not disregardedAlthough we were not able to explain this unexpected resultdevelopment of organisms capable of metabolizing mercuryis possible Several common bacterial genera (EscherichiaEnterobacter and Bacillus) are able to resist HgCl
2through
mercury methylation [41]This has compromised the evalua-tion of the photo-degradation alone and needs to be criticallyconsidered
An important point of our study is that we were able tofind significant fading of DOM only in the light treatmentof the bacterial fraction which suggests the existence of acomplementary pathway of dissolved organic degradation bybacteria and light [13] Farjalla et al [11] also found fading(in terms of 250 365 and 430 nm absorbance) of DOM butdecreased bacterial density and production of bacteria inUV-exposed macrophyte leachates indicating that fading itselfsays little about the bioavailability of compounds formedTheincrease in bacterial density together with the stability of theDOC and Abs250 365 suggests that the photodegradationof high-molecular-weight compounds may have producedassimilable substances for bacterial growth [9 10] but theywere readily consumed by bacteria Another important ques-tion still concerning mercury inside control bags is thatthis metal shows great affinity with organic matter alteringthe conformation of the latter [42] other bivalent heavymetals like Cu+2 Pb+2 and Cd+2 have been found to changephotochemistry of yellow substances and humic acids [43]and it is possible that mercury has had some unevaluatednegative effect on photochemical reactivity in the bags whereit was added to
Simultaneous irradiation and incubation in our studymay have exposed bacteria to continuous production ofinhibitory free radicals as suggested by the decrease inbacterial production Many studies that dealt with bacterialutilization of photochemically transformed organic carbonderived from macrophytes employed an approach of pre-treating the leachates with UV light prior to the incubationof bacterial batches [11 13 44] In many cases this priorexposure has been found to inhibit bacterial production anddecrease growth efficiency at least for a short period whichhas been attributed to the formation of hydrogen peroxideand possibly other free radicals by light [11 45] Formationof nonassimilable compounds is also possible Seitzingeret al [46] studied bacterial utilization of dissolved organicmatter in two streams and found that BP increased greatlyduring two days of incubation while DOC concentrationsignificantly decreased (45ndash50) in 12 days However 60of the compounds found in the complex mixture of organicmolecules were not consumed which Seitzinger et al [46]
6 Journal of Ecosystems
Table2Differencesb
etweeninitialcond
ition
andexpo
sition(light)or
not(dark)o
fmacroph
yte-deriv
edDOM
tosunlight
inun
filteredwater
(Bulk)filteredwater
(Bacteria
l)andCon
trol
(HgC
l 2)for120
hun
derinsitucond
ition
sinthesouthern
partof
thesubtropicalshallo
wlake
Mangueira
(sou
thernBrazil)
teste
dby
one-way
ANOVA
andTu
keyrsquos
test(m
eanplusmnsta
ndard
deviation)
Experim
ent
(I)B
ulk
(II)Ba
cterial
(III)C
ontro
lInitial
Light
Dark
Initial
Light
Dark
Initial
Light
Dark
Bacteriald
ensity
(cells10
6mLminus
1 )081plusmn016
a076plusmn050
a051plusmn041
a17
1plusmn031
a265plusmn009
b063plusmn007
c14
3plusmn013
a10
3plusmn010
a218plusmn033
b
Bacterialbiovolume
(120583m
3 )006plusmn002
a005plusmn001
a005plusmn001
a010plusmn001
a008plusmn001
a006plusmn001
a006plusmn001
a007plusmn002
a007plusmn003
a
Bacterialbiomass
(ngC
mLminus
1 )65plusmn026
a372plusmn206
a333plusmn320
a1864plusmn591
a2473plusmn277
a414plusmn045
b95
7plusmn13
1a807plusmn18
5a1649plusmn357
b
Bacterialprodu
ction
(120583gC
Lminus1 hminus1 )
012plusmn004
a028plusmn024
a015plusmn003
a015plusmn001
a002plusmn002
b014plusmn002
a003plusmn001
a003plusmn002
a077plusmn020
b
Bacterialrespiratio
n(120583gC
Lminus1 hminus1 )
NA
NA
109plusmn012
aNA
NA
228plusmn088
aNA
NA
214plusmn12
7a
BacterialG
rowth
Efficiency
NA
NA
011plusmn002
aNA
NA
007plusmn003
aNA
NA
022plusmn012
a
Ciliatedensity
(times10
4cells
mminus3 )
414plusmn18
0a18
0plusmn312
b1560plusmn178a
0plusmn0
0plusmn0
0plusmn0
0plusmn0
0plusmn0
0plusmn0
Diss
olvedOrganicCa
rbon
(mgC
Lminus1 )
1869plusmn087
a1946plusmn112
a2626plusmn78
3a2507plusmn136
a4358plusmn255
a3534plusmn204
a2090plusmn354
a3575plusmn367
a46
10plusmn175a
Abs430
(Abs
430n
m(times10minus2))
251plusmn022
a221plusmn012
a263plusmn019
a269plusmn006
a19
8plusmn014
b235plusmn020
a244plusmn016
ab18
4plusmn035
b271plusmn025
a
Abs250
365
nmratio
085plusmn009
a085plusmn002
a085plusmn004
a076plusmn007
a079plusmn018
a087plusmn002
a075plusmn005
a079plusmn025
a087plusmn006
a
Sign
ificanceo
fone-w
ayANOVA
andTu
keyrsquos
aposte
rioritestatPlt005
isindicatedwith
different
lowercase
superscriptletterswith
ineach
experim
ent(Initialversus
LightversusD
ark)Th
eexceptio
nisbacterial
respira
tionandgrow
theffi
ciencyw
here
differences
weretestedam
ongexperim
ents
NAn
otavailableb
ecause
respira
tionwas
measuredin
dark
bottlesFor
allvariables
n=3replicates
Journal of Ecosystems 7
suggested may have resulted from some inhibition factorinvolved in their utilization Contrastingly Perez et al [12]exposed natural river-water DOM to light also in a simulta-neous exposure and found bacterial enhancement with UVexposure These differences could be due to variations inorigin age and biochemical composition of the DOM [45]
The generally low bioavailability of DOC in the formof humic substances [6] indicates that a large proportionof the dissolved carbon in Lake Mangueira is refractory tobacterial consumption This Abs250 365 ratio is higher inthe littoral zone than in the pelagic zone suggesting higherproportion of low-molecular-weight (LMW Abs 250 nm)substances in the littoral zone when compared to the pelagiczone In fact this was confirmed (pelagic Abs250 mean= 003 standard deviation = 0002 F(14) = 1163 119875 lt0001 littoral zone data obtained from Table 1) In spiteof the smaller size of their molecules these substances canbe less reactive Amon and Benner [4] conducted a cross-environment experiment where they analyzed the reactivityof low-(LMW lt1 KDa) and high-molecular-weight (HMWgt1 KDa) dissolved compounds and found significant changesin BP and BR during the experiment (also five days)Unexpectedly these bacterial variables were higher in HMWsubstrates and based on these results the authors proposedthe size-reactivity continuum model that predicts that themajor path of degradation goes from large highly reactiveto small highly recalcitrant molecules If this hypothesis isbroadly applicable to many ecosystems it suggests that inLakeMangueira the compounds lixiviated by themacrophytebelt had already degraded to some extent and accumulatedas dissolved unreactive LMW compounds with very lowdegradation rates in the littoral zones
The age of the DOM subjected to bacterial degradationcan be fundamental for its availability In our experiment wefocused on the subsequent stage of macrophyte degradationthat is after bacterial and light reworking had alreadyoccurred inside the macrophyte stands Most studies thatinvestigated microbial degradation of macrophytes focusedon the early stages of degradation with exposure of coarsefragments of macrophytes to leaching For example Wehret al [47] found a 45-fold increase in bacterial productionafter 168 h of incubation of macrophyte detritus ConverselyHolm-Hansen et al [48] demonstrated very low rates ofmicrobial degradation of the emergent macrophyte Juncuseffusus only 23of the total biomass in leaf litter bagswas lostafter 268 days Enhancement of bacterial production mightbe related to the early stage of degradation given that Kuehnet al [49] employed senescent leaves in their experiment asituation more similar to ours
One possible explanation for the low photodegradationrates may be the total amount of UV accumulated during theexperiment In the study of Farjalla et al [11] macrophyteleachates were photochemically transformed with total UVenergy asymp5670KJmminus2 which was able to induce a bacterialpositive response This UV intensity is almost 100 timeshigher than that estimated for the field conditions in thepresent study (asymp58KJmminus2) One implication is that underlaboratory conditions the rates of photo-degradation may
be overestimated compared to natural conditions Anotherimportant point however is that polyethylene bags used inthis study may present a reduced rate of UV transmittance(eg 68 of UVB at 300 nm) [48] Even though the trans-mittance increases slightly towards larger wavelengths [26]this quenching may have imposed slightly reduced rates ofphoto-degradation than in the field
We attempted to determine the importance of the twomain routes of carbon degradation in aquatic systemsthat is bacterial and photo-degradation on macrophyte-derived DOM flowing into Lake Mangueira from a largewetland belt Remarkably our results showed few changesin environmental parameters related to carbon degradation(DOC Abs250 365 and respiration) even though bacterialvariables did show changes under lightdark incubationconditions (120 h) This DOM that enters the lake seemsto be highly unreactive in a short-term and may be thecause for the difference in bacterial metabolism between thelittoral and pelagic zones reported by Ng et al [17] sincethis is the main carbon source for bacteria in the littoralzone It is important to take into consideration that it wasthe first approach to the subject in this lake and was aonetime experiment hence more experimental replicationis needed Also there is a need for testing bacterial growthin dilution cultures nutrients amendment effects of light onrespiration and the size and quality spectra of DOM derivedfrom macrophytes and phytoplankton to better address thissubject Another more speculative hypothesis is a possibleallelopathic effect of macrophytes (mainly submersed) sincethis effect on cyanobacteria is largely known [50 51]This hasbeen considered as a possible explanation for lower bacterialdiversity [52] and metabolism [17] in lake zones extensivelycolonized by macrophytes Direct effects on heterotrophicbacteria however still need to be properly addressed
5 Conclusions
In this study we reported a short-term unchange in macro-phyte-derived DOC availability with exposition to sunlightThis suggests that bacterial and photo-degradation can bevery low under ldquoin siturdquo conditions which presents lowerlight intensity when compared to laboratory experimentsbut more evidence is needed to determine if this is apermanent condition of the system This study adds animportant mechanistic explanation (low reactivity) for theprevious finding that bacterial respiration is lower in littoralwhen compared to pelagic zones in lake Mangueira [53] Wehypothesize that given the climatic conditions continuousgrowth of macrophytes and loading of dissolved recalcitrantcompounds contribute to very slow rates ofDOCdegradationand in a short term slight influence of photo-degradationSubmersed and emergent macrophytes cover extensive areasinside and around the drainage channel where we obtainedthe water for the present experiment and similar extensivemacrophyte coverage is common in many lakes across theworld The acknowledgment and further investigation onthis subject are essential for the understanding of carboncycling and metabolism in systems that are densely coveredby macrophytes
8 Journal of Ecosystems
Conflict of Interests
The authors state no conflict of interests involving any step ofthis study
Acknowledgments
The authors draw particular attention to the assistance pro-vided by theChicoMendes Institute of Biodiversity (ICMBio-ESEC TAIM) Also they sincerely thank Dr Laura Utz forhelping with ciliate sampling and handling and Dr LuizKucharski for providing the scintillation counter They thankthe Conselho Nacional de Desenvolvimento Cientıfico eTecnologico of Brazil (CNPq-Long TermEcological ResearchProgram Site 7 Sistema Hidrologico do Taim) for financialsupport They are also grateful to Dr Janet W Reid (JWRAssociates) for revising the English text
References
[1] R G Wetzel ldquoGradient-dominated ecosystems sources andregulatory functions of dissolved organic matter in freshwaterecosystemsrdquo Hydrobiologia vol 229 no 1 pp 181ndash198 1992
[2] G H Lauster P C Hanson and T K Kratz ldquoGross primaryproduction and respiration differences among littoral andpelagic habitats in northernWisconsin lakesrdquoCanadian Journalof Fisheries and Aquatic Sciences vol 63 no 5 pp 1130ndash11412006
[3] K M Docherty K C Young P A Maurice and S DBridgham ldquoDissolved organicmatter concentration and qualityinfluences upon structure and function of freshwater microbialcommunitiesrdquo Microbial Ecology vol 52 no 3 pp 378ndash3882006
[4] R M W Amon and R Benner ldquoBacterial utilization ofdifferent size classes of dissolved organicmatterrdquo Limnology andOceanography vol 41 no 1 pp 41ndash51 1996
[5] L Bracchini A Cozar AM Dattilo et al ldquoThe role of wetlandsin the chromophoric dissolved organic matter release and itsrelation to aquatic ecosystems optical properties A case ofstudy katonga and Bunjako Bays (Victoria Lake Uganda)rdquoChemosphere vol 63 no 7 pp 1170ndash1178 2006
[6] U Munster and R J Chrost ldquoOrigin composition and micro-bial utilization of dissolved organicmatterrdquo inAquaticMicrobialEcology J Overbeck and R J Chrost Eds pp 8ndash46 SpringerNew York NY USA 1990
[7] D O Hessen ldquoDissolved organic carbon in a humic lake effectson bacterial production and respirationrdquo Hydrobiologia vol229 no 1 pp 115ndash123 1992
[8] M J Lindell W Graneli and L J Tranvik ldquoEnhanced bac-terial growth in response to photochemical transformation ofdissolved organicmatterrdquo Limnology andOceanography vol 40no 1 pp 195ndash199 1995
[9] M A Moran and R G Zepp ldquoRole of photoreactions inthe formation of biologically labile compounds from dissolvedorganicmatterrdquo Limnology and Oceanography vol 42 no 6 pp1307ndash1316 1997
[10] S Bertilsson and L J Tranvik ldquoPhotochemically producedcarboxylic acids as substrates for freshwater bacterioplanktonrdquoLimnology and Oceanography vol 43 no 5 pp 885ndash895 1998
[11] V F Farjalla A M Anesio S Bertilsson and W GranelildquoPhotochemical reactivity of aquatic macrophyte leachates abi-otic transformations and bacterial responserdquo Aquatic MicrobialEcology vol 24 no 2 pp 187ndash195 2001
[12] A P Perez M M Diaz M A Ferraro G C Cusminsky andH E Zagarese ldquoReplicated mesocosm study on the role ofnatural ultraviolet radiation in high CDOM shallow lakesrdquoPhotochemical and Photobiological Sciences vol 2 no 2 pp 118ndash123 2003
[13] A M Amado V F Farjalla F D A Esteves R L BozelliF Roland and A Enrich-Prast ldquoComplementary pathwaysof dissolved organic carbon removal pathways in clear-waterAmazonian ecosystems photochemical degradation and bacte-rial uptakerdquo FEMSMicrobiology Ecology vol 56 no 1 pp 8ndash172006
[14] S G Ribblett M A Palmer and D W Coats ldquoThe importanceof bacterivorous protists in the decomposition of stream leaflitterrdquo Freshwater Biology vol 50 no 3 pp 516ndash526 2005
[15] R A Snyder and M P Hoch ldquoConsequences of protist-stimulated bacterial production for estimating protist growthefficienciesrdquo Hydrobiologia vol 341 no 2 pp 113ndash123 1996
[16] N Rooney and J Kalff ldquoInteractions among epilimneticphosphorus phytoplankton biomass and bacterioplanktonmetabolism in lakes of varying submerged macrophyte coverrdquoHydrobiologia vol 501 pp 75ndash81 2003
[17] H-T Ng D da Motta Marques E Jeppesen and MSoslashndergaard ldquoBacterioplankton in the littoral and pelagic zonesof subtropical shallow lakesrdquo Hydrobiologia vol 646 no 1 pp311ndash326 2010
[18] L O Crossetti L S Cardoso V LM Callegaro et al ldquoInfluenceof the hydrological changes on the phytoplankton structureand dynamics in a subtropical wetland-lake systemrdquo ActaLimnologica Brasiliensia vol 19 pp 315ndash329 2007
[19] C R Fragoso Jr D M L M Marques W Collischonn C EM Tucci and E H vanNes ldquoModelling spatial heterogeneity ofphytoplankton in Lake Mangueira a large shallow subtropicallake in South Brazilrdquo Ecological Modelling vol 219 no 1-2 pp125ndash137 2008
[20] J C Ceballos and M L Rodrigues ldquoEstimativa de insolacaomediante satelite geoestacionario resultados preliminaresrdquo inProceedings of 15th Congresso Brasileiro de Meteorologia INPESao Paulo Brazil 2008
[21] R G Wetzel and G E Likens Limnological Analyses SpringerNew York NY USA 3rd edition 2000
[22] American Public Health Association American Water WorksAssociation and Water Environment Federation StandardMethods For the Examination of Water and Wastewater Amer-ican Public Health Associatio Washington DC USA 20thedition 1999
[23] F J H Mackereth J Heron and J F Talling Water AnalysisSome Revised Methods For Limnologists Freshwater BiologicalAssociation Ambleside UK 2nd edition 1989
[24] D J Strome and M C Miller ldquoPhotolytic changes in dissolvedhumic substancesrdquo Verhandlungen der Internationalen Vereini-gung fur Theoretische und Angewandte Limnologie vol 20 pp1248ndash1254 1978
[25] J R Helms A Stubbins J D Ritchie E C Minor D J Kieberand K Mopper ldquoAbsorption spectral slopes and slope ratiosas indicators of molecular weight source and photobleachingof chromophoric dissolved organic matterrdquo Limnology andOceanography vol 53 no 3 pp 955ndash969 2008
Journal of Ecosystems 9
[26] P AasMM Lyons R Pledger D LMitchell andWH JeffreyldquoInhibition of bacterial activities by solar radiation in nearshorewaters and the Gulf of Mexicordquo Aquatic Microbial Ecology vol11 no 3 pp 229ndash238 1996
[27] R Massana J M Gasol P K Bjoslashrnsen et al ldquoMeasurement ofbacterial size via image analysis of epifluorescence preparationsdescription of an inexpensive system and solutions to some ofthe most common problemsrdquo Scientia Marina vol 61 no 3 pp397ndash407 1997
[28] R L J R Kepner Jr and J R Pratt ldquoUse of fluorochromes fordirect enumeration of total bacteria in environmental samplespast and presentrdquo Microbiological Reviews vol 58 no 4 pp603ndash615 1994
[29] J Liu F B Dazzo O Glagoleva B Yu andA K Jain ldquoCMEIASa computer-aided system for the image analysis of bacterialmorphotypes in microbial communitiesrdquo Microbial Ecologyvol 41 no 3 pp 173ndash194 2001
[30] S Norland ldquoThe relationship between biomass and volume ofbacteriardquo inHandbook of Methods in Aquatic Microbial EcologyP F Kemp B F Sherr E B Sherr and J J Cole Eds pp 339ndash345 Lewis Chelsea Mich USA 1993
[31] D C Simon and F Azam ldquoA simple economical method formeasuring bacterial protein synthesis rates in sea water using3H-leucinerdquo Marine Microbial Food Webs vol 6 pp 107ndash1091992
[32] D Kirchman ldquoMeasuring bacterial biomass production andgrowth rates from leucine incorporation in natural aquatic envi-ronmentsrdquo in MarIne MicrobiologymdashMethods In MicrobiologyJ H Paul Ed pp 227ndash237 Academic Press San Diego CalifUSA 30th edition 2001
[33] H L Golterman R S Clymo and M A M OhnstadMethodsfor Physical and Chemical Analysis of Fresh Waters BlackwellPublishing Company London UK 2nd edition 1978
[34] P Del Giorgio ldquoRapid and precise determination of dissolvedoxygen by spectrophotometry evaluation of interference fromcolor and turbidityrdquo Limnology and Oceanography vol 44 no4 pp 1148ndash1154 1999
[35] P A del Giorgio J J Cole and A Cimbleris ldquoRespiration ratesin bacteria exceed phytoplankton production in unproductiveaquatic systemsrdquo Nature vol 385 no 6612 pp 148ndash151 1997
[36] P A del Giorgio and J J Cole ldquoBacterial growth efficiencyin natural aquatic systemsrdquo Annual Review of Ecology andSystematics vol 29 pp 503ndash541 1998
[37] RDevelopmentCoreTeamRALanguage andEnvironment ForStatistical Computing R Foundation for Statistical ComputingVienna Austria 2011 httpwwwR-projectorg
[38] H J de Lange D P Morris and C E Williamson ldquoSolarultraviolet photodegradation of DOCmay stimulate freshwaterfood websrdquo Journal of Plankton Research vol 25 no 1 pp 111ndash117 2003
[39] K Kalinowska ldquoBacteria nanoflagellates and ciliates as com-ponents of the microbial loop in three lakes of different trophicstatusrdquo Polish Journal of Ecology vol 52 no 1 pp 19ndash34 2004
[40] M A Gates ldquoQuantitative importance of ciliates in the plank-tonic biomass of lake ecosystemsrdquoHydrobiologia vol 108 no 3pp 233ndash238 1984
[41] M K Hamdy and O R Noyes ldquoFormation of methyl mercuryby bacteriardquo Journal of Applied Microbiology vol 30 no 3 pp424ndash432 1975
[42] MRavichandran ldquoInteractions betweenmercury and dissolvedorganic mattermdasha reviewrdquo Chemosphere vol 55 no 3 pp 319ndash331 2004
[43] G M Ferrari ldquoInfluence of pH and heavy metals in thedetermination of yellow substance in estuarine areasrdquo RemoteSensing of Environment vol 37 no 2 pp 89ndash100 1991
[44] A M Anesio J Theil-Nielsen and W Graneli ldquoBacterialgrowth on photochemically transformed leachates from aquaticand terrestrial primary producersrdquo Microbial Ecology vol 40no 3 pp 200ndash208 2000
[45] A M Anesio W Graneli G R Aiken D J Kieber andK Mopper ldquoEffect of humic substance photodegradation onbacterial growth and respiration in lake waterrdquo Applied andEnvironmentalMicrobiology vol 71 no 10 pp 6267ndash6275 2005
[46] S P Seitzinger H Hartnett R Lauck et al ldquoMolecular-level chemical characterization and bioavailability of dissolvedorganic matter in stream water using electrospray-ionizationmass spectrometryrdquo Limnology and Oceanography vol 50 no1 pp 1ndash12 2005
[47] J D Wehr J Petersen and S Findlay ldquoInfluence of threecontrasting detrital carbon sources on planktonic bacterialmetabolism in a mesotrophic lakerdquo Microbial Ecology vol 37no 1 pp 23ndash35 1999
[48] O Holm-Hansen E W Helbling B B Prezelin and RC Smith ldquoPolyethylene bags and solar ultraviolet radiationrdquoScience vol 259 no 5094 pp 534ndash535 1993
[49] K A Kuehn M J Lemke K Suberkropp and R G WetzelldquoMicrobial biomass and production associated with decayingleaf litter of the emergent macrophyte Juncus effususrdquo Limnol-ogy and Oceanography vol 45 no 4 pp 862ndash870 2000
[50] E M Gross S Hilt P Lombardo and G Mulderij ldquoSearch-ing for allelopathic effects of submerged macrophytes onphytoplanktonmdashstate of the art and open questionsrdquo Hydrobi-ologia vol 584 no 1 pp 77ndash88 2007
[51] G Mulderij E H van Nes and E van Donk ldquoMacrophyte-phytoplankton interactions the relative importance of allelopa-thy versus other factorsrdquo Ecological Modelling vol 204 no 1-2pp 85ndash92 2007
[52] Q L Wu G Zwart J Wu M P Kamst-van Agterveld S Liuand M W Hahn ldquoSubmersed macrophytes play a key role instructuring bacterioplankton community composition in thelarge shallow subtropical Taihu Lake Chinardquo EnvironmentalMicrobiology vol 9 no 11 pp 2765ndash2774 2007
[53] N HThey D M Marques and R S Souza ldquoLower respirationin the littoral zone of a subtropical shallow lakerdquo Frontiers inMicrobiology vol 3 p 434 2012
sample (ng C mLminus1) Bacterial production rates (BP) wereestimated through the method of (L[45-3H]) radiolabeledleucine microcentrifugation Incubations for bacterial pro-ductionwere carried out in Eppendorf vials (2mL) and lasted30min inside a tray filledwith lakewater at room temperature(15-16∘C) Radioactivity counts were made after addition of1mL of scintillation liquid (Optiphase HiSafe III Wallac)in a Rack Beta Liquid Scintillation Counter (LKB Wallac1209 Massachusetts USA) for 180 s twice The calculationsassumed the isotopic dilution to be equal to 2 the molar ratioof leucine in the protein pool to be 0073 and the ratio ofcarbonprotein contents to be 0086 [31 32]
The bacterial respiration in the fractions (bulk bacterialand control) was estimated as the rate of oxygen consump-tion through the Winkler method [33] with colorimetricreading [34] This method was conducted separately througha parallel incubation of the fractions in glass BOD bottles of100mL capacity Incubation was done in the dark (double-wrapping the bottles with aluminum foil) with a total of 18bottles (9 initial bottles and 9 final bottles three triplicatesof each of the three fractions) and then only comparisonsamong fractions could be made The bottles were tied bytheir necks to another floating PVC pipe square that wasanchored next to the incubating bags For calculations eachfinal value of O
2was subtracted from the mean of the three
initial bottles and divided by the incubation time assumingconstant respiration rate over time The molar conversionfactor between carbon and oxygen was assumed to be equalto 10 [35]
Bacterial GrowthEfficiency (BGE)was computed accord-ing to equation (I) of del Giorgio et al [36] (I) BGE =(BP)(BP + BR) where BP is the bacterial production rateand BR is the bacterial respiration rate Because respirationwas recorded only for the three fractions also BGE couldonly be calculated and compared among fractions For thecalculations in order to match the results from respirationwe employed the mean of the bacterial production ratebetween the initial and final incubations
Ciliates Abs430 and Abs250 365 were measured thesame way as for field samples [8 21 24]
25 Statistical Treatment Differences in the variables (bacte-rial density biovolume biomass production ciliate densityDOC Abs 430 nm and Abs250 365 nm ratio) among initiallight and dark treatments in each fraction were tested withone-way ANOVA using R 2131 [37] Significant differencesrevealed by ANOVA were tested by Tukeyrsquos a posteriori testIncreases or decreases refer to the light and dark treatmentsin relation to the respective initial condition Bacterial respi-ration and BGE were tested for differences among fractionsand field condition through ANOVA and Tukeyrsquos test
3 Results
The main limnological variables for the experimental condi-tions can be found in Table 1The global cumulative radiation(GR) during the experiment was asymp25 455KJmminus2 Total UVradiation received was estimated to be asymp58 kJmminus2
Bacterial density did not differ among treatments in thebulk fraction it increased with light exposure and decreasedin the dark bags in the bacterial fraction In the controlfraction there was an increase in density in the dark treat-ment Bacterial biovolume did not change with treatmentsBacterial biomass also showed no differences in the bulkfraction It decreased in the dark treatment for the bacterialfraction and increased in the dark treatment in the controlfraction (Table 2)
Bacterial production was unaltered in the bulk fractionIn the bacterial fraction it decreased in the light treatmentand increased in the dark In the control fraction it increasedin the dark Bacterial respiration and BGE did not differamong the fractions (Table 2)
Comparing the bacterial production with the field con-dition only the dark control treatment showed a significantdifference (ANOVA F(920) = 2941 119875 ≪ 0001 dark controlgt Field 119875 ≪ 0001) Bacterial respiration did not differ fromthe field condition in any treatment (ANOVA F(38) = 2517119875 = 0132) BGE decreased in the bulk and bacterial fractionincubations (ANOVAF(38) = 492119875 = 00318 FieldgtBulk119875 = 0058 Field gt Bacterial 119875 = 0052)
In the bulk fraction total ciliate density changed only inthe light treatmentThe light treatment at its turn decreasedcompared to the initial treatment The dark incubation didnot differ statistically from the initial treatmentThe filtrationprocedure successfully excluded ciliates as no ciliates werefound in the bacterial and control fractions any ciliate inthe control was likely lysed by the toxic inhibitor (Table 2)No flagellates were found in the filtered samples undermicroscopic examination
DOC and Abs250 365 showed no effect of treatmentfor any fraction Abs430 (water color) was unaltered in thebulk fraction for all treatments only the bacterial fractionshowed significant color fading in the light treatment (26lower)The control light bags did not show any alterationwithtreatment but the color was less intense than in the dark bagsRates of fading in the light treatment were 00006 00014and 00012 dayminus1 absorbance units for the bulk bacterial andcontrol fractions respectively (Table 2)
4 Discussion
The littoral zones of Lake Mangueira (especially in thenorth and south) are extensively colonized by emergent andsubmersed macrophytes and these plants are expected tocontribute large amounts of organic carbon to the systemMuch of this carbon enters the lake in the form of DOMwhich we found to show little reactivity (did not undergodecomposition by bacteria or light) during short-term incu-bation under in situ conditions We were unable to detectchanges in DOC Abs250 365 and bacterial respirationalthough the water color faded in the light bags Theseresults indicated that photodegradation did occur but didnot modify carbon availability to bacteria Exposure tolight however had contrasting effects it increased bacterialdensity in the bacterial fraction suggesting a positive effectof light but decreased bacterial production in this same
Journal of Ecosystems 5
Table 1 Limnological and biological characterization of the water in the initial conditions of the experiment
1Mean air temperature for the 5 days of incubation2Mean daily flux of GR during incubation (n = 5) 1W = 1 J sminus1 For all other variables n = 33Figures in parentheses are plusmnstandard deviations4119885mean mean depth (m) AT air temperature (∘C) TS total solids (mg Lminus1) Alk alkalinity (120583Eq Lminus1) TN total nitrogen (120583g Lminus1) TP total phosphorus(120583g Lminus1) DOC dissolved organic carbon (mg Lminus1) Abs430 absorbance at 430 nm GR global radiation flux (Wmminus2) LMW low-molecular-weight substances(absorbance at 250 nm)HMW high-molecular-weight substances (absorbance at 365 nm) Abs250 365 (ratio of absorbances at 250 and 365 nm) BP bacterialproduction (120583gC L hminus1) BR bacterial respiration (120583gC L hminus1) BGE bacterial growth efficiency
treatmentfraction suggesting photoinhibition We did notexamine the effect of exposure to light on respiration but ourresults showed no difference in respiration from a previousmeasurement in the field (Table 1) The decrease in BGEwith incubation can be attributed to the increase in respi-ration (even though there was no significant differences inrespiration among fractions it impacted the BGE calculationbecause respiration tended to be higher in bacterial andcontrol fractions)
The general lack of changes in practically all variables inthe bulk fraction indicates that there may be an interactionof grazing with radiation The increase of bacterial densitywith light found in the bacterial fraction may have beenimpeded by grazing in the bulk fraction Higher densitiesof bacteria and bacterial grazers have been found in UV-irradiated DOC suggesting that photo-degradation acts as astimulus to microbial food webs [38] In our case howeverthe decrease in ciliate density with light treatment in thebulk fraction indicated a net effect of photoinhibition onciliates If they did control bacterial density it was at acost of a higher grazing rate per cell On the other side ithas been found that high concentrations of refractory DOClike in humic lakes may be associated to lower bacterialnanoflagellates and ciliates numbers and biomass and hencea less efficient functioning of the microbial loop that islower rates of DOC reincorporation to the food web [39]The density of ciliates in the initial condition (presumably afield concentration estimate) and inside the bags was low (2ndash4 orders of magnitude) when compared to values reportedfor lakes with variable trophic status where they range from19 to 100 times 106 cellsmminus3 (see compilation in Gates [40])Thissuggests that there may be a minor role of ciliates on thedecomposition rates of this refractory DOC in Mangueiralakersquos littoral zones
Unexpectedly we found enhancement in bacterial den-sity biomass and production in the dark treatment of thecontrol fraction This enhancement was first attributed tosome kind of contamination but since no ciliates werefound in these bags and contamination in the three bagssimultaneously is unlikely this result was not disregardedAlthough we were not able to explain this unexpected resultdevelopment of organisms capable of metabolizing mercuryis possible Several common bacterial genera (EscherichiaEnterobacter and Bacillus) are able to resist HgCl
2through
mercury methylation [41]This has compromised the evalua-tion of the photo-degradation alone and needs to be criticallyconsidered
An important point of our study is that we were able tofind significant fading of DOM only in the light treatmentof the bacterial fraction which suggests the existence of acomplementary pathway of dissolved organic degradation bybacteria and light [13] Farjalla et al [11] also found fading(in terms of 250 365 and 430 nm absorbance) of DOM butdecreased bacterial density and production of bacteria inUV-exposed macrophyte leachates indicating that fading itselfsays little about the bioavailability of compounds formedTheincrease in bacterial density together with the stability of theDOC and Abs250 365 suggests that the photodegradationof high-molecular-weight compounds may have producedassimilable substances for bacterial growth [9 10] but theywere readily consumed by bacteria Another important ques-tion still concerning mercury inside control bags is thatthis metal shows great affinity with organic matter alteringthe conformation of the latter [42] other bivalent heavymetals like Cu+2 Pb+2 and Cd+2 have been found to changephotochemistry of yellow substances and humic acids [43]and it is possible that mercury has had some unevaluatednegative effect on photochemical reactivity in the bags whereit was added to
Simultaneous irradiation and incubation in our studymay have exposed bacteria to continuous production ofinhibitory free radicals as suggested by the decrease inbacterial production Many studies that dealt with bacterialutilization of photochemically transformed organic carbonderived from macrophytes employed an approach of pre-treating the leachates with UV light prior to the incubationof bacterial batches [11 13 44] In many cases this priorexposure has been found to inhibit bacterial production anddecrease growth efficiency at least for a short period whichhas been attributed to the formation of hydrogen peroxideand possibly other free radicals by light [11 45] Formationof nonassimilable compounds is also possible Seitzingeret al [46] studied bacterial utilization of dissolved organicmatter in two streams and found that BP increased greatlyduring two days of incubation while DOC concentrationsignificantly decreased (45ndash50) in 12 days However 60of the compounds found in the complex mixture of organicmolecules were not consumed which Seitzinger et al [46]
6 Journal of Ecosystems
Table2Differencesb
etweeninitialcond
ition
andexpo
sition(light)or
not(dark)o
fmacroph
yte-deriv
edDOM
tosunlight
inun
filteredwater
(Bulk)filteredwater
(Bacteria
l)andCon
trol
(HgC
l 2)for120
hun
derinsitucond
ition
sinthesouthern
partof
thesubtropicalshallo
wlake
Mangueira
(sou
thernBrazil)
teste
dby
one-way
ANOVA
andTu
keyrsquos
test(m
eanplusmnsta
ndard
deviation)
Experim
ent
(I)B
ulk
(II)Ba
cterial
(III)C
ontro
lInitial
Light
Dark
Initial
Light
Dark
Initial
Light
Dark
Bacteriald
ensity
(cells10
6mLminus
1 )081plusmn016
a076plusmn050
a051plusmn041
a17
1plusmn031
a265plusmn009
b063plusmn007
c14
3plusmn013
a10
3plusmn010
a218plusmn033
b
Bacterialbiovolume
(120583m
3 )006plusmn002
a005plusmn001
a005plusmn001
a010plusmn001
a008plusmn001
a006plusmn001
a006plusmn001
a007plusmn002
a007plusmn003
a
Bacterialbiomass
(ngC
mLminus
1 )65plusmn026
a372plusmn206
a333plusmn320
a1864plusmn591
a2473plusmn277
a414plusmn045
b95
7plusmn13
1a807plusmn18
5a1649plusmn357
b
Bacterialprodu
ction
(120583gC
Lminus1 hminus1 )
012plusmn004
a028plusmn024
a015plusmn003
a015plusmn001
a002plusmn002
b014plusmn002
a003plusmn001
a003plusmn002
a077plusmn020
b
Bacterialrespiratio
n(120583gC
Lminus1 hminus1 )
NA
NA
109plusmn012
aNA
NA
228plusmn088
aNA
NA
214plusmn12
7a
BacterialG
rowth
Efficiency
NA
NA
011plusmn002
aNA
NA
007plusmn003
aNA
NA
022plusmn012
a
Ciliatedensity
(times10
4cells
mminus3 )
414plusmn18
0a18
0plusmn312
b1560plusmn178a
0plusmn0
0plusmn0
0plusmn0
0plusmn0
0plusmn0
0plusmn0
Diss
olvedOrganicCa
rbon
(mgC
Lminus1 )
1869plusmn087
a1946plusmn112
a2626plusmn78
3a2507plusmn136
a4358plusmn255
a3534plusmn204
a2090plusmn354
a3575plusmn367
a46
10plusmn175a
Abs430
(Abs
430n
m(times10minus2))
251plusmn022
a221plusmn012
a263plusmn019
a269plusmn006
a19
8plusmn014
b235plusmn020
a244plusmn016
ab18
4plusmn035
b271plusmn025
a
Abs250
365
nmratio
085plusmn009
a085plusmn002
a085plusmn004
a076plusmn007
a079plusmn018
a087plusmn002
a075plusmn005
a079plusmn025
a087plusmn006
a
Sign
ificanceo
fone-w
ayANOVA
andTu
keyrsquos
aposte
rioritestatPlt005
isindicatedwith
different
lowercase
superscriptletterswith
ineach
experim
ent(Initialversus
LightversusD
ark)Th
eexceptio
nisbacterial
respira
tionandgrow
theffi
ciencyw
here
differences
weretestedam
ongexperim
ents
NAn
otavailableb
ecause
respira
tionwas
measuredin
dark
bottlesFor
allvariables
n=3replicates
Journal of Ecosystems 7
suggested may have resulted from some inhibition factorinvolved in their utilization Contrastingly Perez et al [12]exposed natural river-water DOM to light also in a simulta-neous exposure and found bacterial enhancement with UVexposure These differences could be due to variations inorigin age and biochemical composition of the DOM [45]
The generally low bioavailability of DOC in the formof humic substances [6] indicates that a large proportionof the dissolved carbon in Lake Mangueira is refractory tobacterial consumption This Abs250 365 ratio is higher inthe littoral zone than in the pelagic zone suggesting higherproportion of low-molecular-weight (LMW Abs 250 nm)substances in the littoral zone when compared to the pelagiczone In fact this was confirmed (pelagic Abs250 mean= 003 standard deviation = 0002 F(14) = 1163 119875 lt0001 littoral zone data obtained from Table 1) In spiteof the smaller size of their molecules these substances canbe less reactive Amon and Benner [4] conducted a cross-environment experiment where they analyzed the reactivityof low-(LMW lt1 KDa) and high-molecular-weight (HMWgt1 KDa) dissolved compounds and found significant changesin BP and BR during the experiment (also five days)Unexpectedly these bacterial variables were higher in HMWsubstrates and based on these results the authors proposedthe size-reactivity continuum model that predicts that themajor path of degradation goes from large highly reactiveto small highly recalcitrant molecules If this hypothesis isbroadly applicable to many ecosystems it suggests that inLakeMangueira the compounds lixiviated by themacrophytebelt had already degraded to some extent and accumulatedas dissolved unreactive LMW compounds with very lowdegradation rates in the littoral zones
The age of the DOM subjected to bacterial degradationcan be fundamental for its availability In our experiment wefocused on the subsequent stage of macrophyte degradationthat is after bacterial and light reworking had alreadyoccurred inside the macrophyte stands Most studies thatinvestigated microbial degradation of macrophytes focusedon the early stages of degradation with exposure of coarsefragments of macrophytes to leaching For example Wehret al [47] found a 45-fold increase in bacterial productionafter 168 h of incubation of macrophyte detritus ConverselyHolm-Hansen et al [48] demonstrated very low rates ofmicrobial degradation of the emergent macrophyte Juncuseffusus only 23of the total biomass in leaf litter bagswas lostafter 268 days Enhancement of bacterial production mightbe related to the early stage of degradation given that Kuehnet al [49] employed senescent leaves in their experiment asituation more similar to ours
One possible explanation for the low photodegradationrates may be the total amount of UV accumulated during theexperiment In the study of Farjalla et al [11] macrophyteleachates were photochemically transformed with total UVenergy asymp5670KJmminus2 which was able to induce a bacterialpositive response This UV intensity is almost 100 timeshigher than that estimated for the field conditions in thepresent study (asymp58KJmminus2) One implication is that underlaboratory conditions the rates of photo-degradation may
be overestimated compared to natural conditions Anotherimportant point however is that polyethylene bags used inthis study may present a reduced rate of UV transmittance(eg 68 of UVB at 300 nm) [48] Even though the trans-mittance increases slightly towards larger wavelengths [26]this quenching may have imposed slightly reduced rates ofphoto-degradation than in the field
We attempted to determine the importance of the twomain routes of carbon degradation in aquatic systemsthat is bacterial and photo-degradation on macrophyte-derived DOM flowing into Lake Mangueira from a largewetland belt Remarkably our results showed few changesin environmental parameters related to carbon degradation(DOC Abs250 365 and respiration) even though bacterialvariables did show changes under lightdark incubationconditions (120 h) This DOM that enters the lake seemsto be highly unreactive in a short-term and may be thecause for the difference in bacterial metabolism between thelittoral and pelagic zones reported by Ng et al [17] sincethis is the main carbon source for bacteria in the littoralzone It is important to take into consideration that it wasthe first approach to the subject in this lake and was aonetime experiment hence more experimental replicationis needed Also there is a need for testing bacterial growthin dilution cultures nutrients amendment effects of light onrespiration and the size and quality spectra of DOM derivedfrom macrophytes and phytoplankton to better address thissubject Another more speculative hypothesis is a possibleallelopathic effect of macrophytes (mainly submersed) sincethis effect on cyanobacteria is largely known [50 51]This hasbeen considered as a possible explanation for lower bacterialdiversity [52] and metabolism [17] in lake zones extensivelycolonized by macrophytes Direct effects on heterotrophicbacteria however still need to be properly addressed
5 Conclusions
In this study we reported a short-term unchange in macro-phyte-derived DOC availability with exposition to sunlightThis suggests that bacterial and photo-degradation can bevery low under ldquoin siturdquo conditions which presents lowerlight intensity when compared to laboratory experimentsbut more evidence is needed to determine if this is apermanent condition of the system This study adds animportant mechanistic explanation (low reactivity) for theprevious finding that bacterial respiration is lower in littoralwhen compared to pelagic zones in lake Mangueira [53] Wehypothesize that given the climatic conditions continuousgrowth of macrophytes and loading of dissolved recalcitrantcompounds contribute to very slow rates ofDOCdegradationand in a short term slight influence of photo-degradationSubmersed and emergent macrophytes cover extensive areasinside and around the drainage channel where we obtainedthe water for the present experiment and similar extensivemacrophyte coverage is common in many lakes across theworld The acknowledgment and further investigation onthis subject are essential for the understanding of carboncycling and metabolism in systems that are densely coveredby macrophytes
8 Journal of Ecosystems
Conflict of Interests
The authors state no conflict of interests involving any step ofthis study
Acknowledgments
The authors draw particular attention to the assistance pro-vided by theChicoMendes Institute of Biodiversity (ICMBio-ESEC TAIM) Also they sincerely thank Dr Laura Utz forhelping with ciliate sampling and handling and Dr LuizKucharski for providing the scintillation counter They thankthe Conselho Nacional de Desenvolvimento Cientıfico eTecnologico of Brazil (CNPq-Long TermEcological ResearchProgram Site 7 Sistema Hidrologico do Taim) for financialsupport They are also grateful to Dr Janet W Reid (JWRAssociates) for revising the English text
References
[1] R G Wetzel ldquoGradient-dominated ecosystems sources andregulatory functions of dissolved organic matter in freshwaterecosystemsrdquo Hydrobiologia vol 229 no 1 pp 181ndash198 1992
[2] G H Lauster P C Hanson and T K Kratz ldquoGross primaryproduction and respiration differences among littoral andpelagic habitats in northernWisconsin lakesrdquoCanadian Journalof Fisheries and Aquatic Sciences vol 63 no 5 pp 1130ndash11412006
[3] K M Docherty K C Young P A Maurice and S DBridgham ldquoDissolved organicmatter concentration and qualityinfluences upon structure and function of freshwater microbialcommunitiesrdquo Microbial Ecology vol 52 no 3 pp 378ndash3882006
[4] R M W Amon and R Benner ldquoBacterial utilization ofdifferent size classes of dissolved organicmatterrdquo Limnology andOceanography vol 41 no 1 pp 41ndash51 1996
[5] L Bracchini A Cozar AM Dattilo et al ldquoThe role of wetlandsin the chromophoric dissolved organic matter release and itsrelation to aquatic ecosystems optical properties A case ofstudy katonga and Bunjako Bays (Victoria Lake Uganda)rdquoChemosphere vol 63 no 7 pp 1170ndash1178 2006
[6] U Munster and R J Chrost ldquoOrigin composition and micro-bial utilization of dissolved organicmatterrdquo inAquaticMicrobialEcology J Overbeck and R J Chrost Eds pp 8ndash46 SpringerNew York NY USA 1990
[7] D O Hessen ldquoDissolved organic carbon in a humic lake effectson bacterial production and respirationrdquo Hydrobiologia vol229 no 1 pp 115ndash123 1992
[8] M J Lindell W Graneli and L J Tranvik ldquoEnhanced bac-terial growth in response to photochemical transformation ofdissolved organicmatterrdquo Limnology andOceanography vol 40no 1 pp 195ndash199 1995
[9] M A Moran and R G Zepp ldquoRole of photoreactions inthe formation of biologically labile compounds from dissolvedorganicmatterrdquo Limnology and Oceanography vol 42 no 6 pp1307ndash1316 1997
[10] S Bertilsson and L J Tranvik ldquoPhotochemically producedcarboxylic acids as substrates for freshwater bacterioplanktonrdquoLimnology and Oceanography vol 43 no 5 pp 885ndash895 1998
[11] V F Farjalla A M Anesio S Bertilsson and W GranelildquoPhotochemical reactivity of aquatic macrophyte leachates abi-otic transformations and bacterial responserdquo Aquatic MicrobialEcology vol 24 no 2 pp 187ndash195 2001
[12] A P Perez M M Diaz M A Ferraro G C Cusminsky andH E Zagarese ldquoReplicated mesocosm study on the role ofnatural ultraviolet radiation in high CDOM shallow lakesrdquoPhotochemical and Photobiological Sciences vol 2 no 2 pp 118ndash123 2003
[13] A M Amado V F Farjalla F D A Esteves R L BozelliF Roland and A Enrich-Prast ldquoComplementary pathwaysof dissolved organic carbon removal pathways in clear-waterAmazonian ecosystems photochemical degradation and bacte-rial uptakerdquo FEMSMicrobiology Ecology vol 56 no 1 pp 8ndash172006
[14] S G Ribblett M A Palmer and D W Coats ldquoThe importanceof bacterivorous protists in the decomposition of stream leaflitterrdquo Freshwater Biology vol 50 no 3 pp 516ndash526 2005
[15] R A Snyder and M P Hoch ldquoConsequences of protist-stimulated bacterial production for estimating protist growthefficienciesrdquo Hydrobiologia vol 341 no 2 pp 113ndash123 1996
[16] N Rooney and J Kalff ldquoInteractions among epilimneticphosphorus phytoplankton biomass and bacterioplanktonmetabolism in lakes of varying submerged macrophyte coverrdquoHydrobiologia vol 501 pp 75ndash81 2003
[17] H-T Ng D da Motta Marques E Jeppesen and MSoslashndergaard ldquoBacterioplankton in the littoral and pelagic zonesof subtropical shallow lakesrdquo Hydrobiologia vol 646 no 1 pp311ndash326 2010
[18] L O Crossetti L S Cardoso V LM Callegaro et al ldquoInfluenceof the hydrological changes on the phytoplankton structureand dynamics in a subtropical wetland-lake systemrdquo ActaLimnologica Brasiliensia vol 19 pp 315ndash329 2007
[19] C R Fragoso Jr D M L M Marques W Collischonn C EM Tucci and E H vanNes ldquoModelling spatial heterogeneity ofphytoplankton in Lake Mangueira a large shallow subtropicallake in South Brazilrdquo Ecological Modelling vol 219 no 1-2 pp125ndash137 2008
[20] J C Ceballos and M L Rodrigues ldquoEstimativa de insolacaomediante satelite geoestacionario resultados preliminaresrdquo inProceedings of 15th Congresso Brasileiro de Meteorologia INPESao Paulo Brazil 2008
[21] R G Wetzel and G E Likens Limnological Analyses SpringerNew York NY USA 3rd edition 2000
[22] American Public Health Association American Water WorksAssociation and Water Environment Federation StandardMethods For the Examination of Water and Wastewater Amer-ican Public Health Associatio Washington DC USA 20thedition 1999
[23] F J H Mackereth J Heron and J F Talling Water AnalysisSome Revised Methods For Limnologists Freshwater BiologicalAssociation Ambleside UK 2nd edition 1989
[24] D J Strome and M C Miller ldquoPhotolytic changes in dissolvedhumic substancesrdquo Verhandlungen der Internationalen Vereini-gung fur Theoretische und Angewandte Limnologie vol 20 pp1248ndash1254 1978
[25] J R Helms A Stubbins J D Ritchie E C Minor D J Kieberand K Mopper ldquoAbsorption spectral slopes and slope ratiosas indicators of molecular weight source and photobleachingof chromophoric dissolved organic matterrdquo Limnology andOceanography vol 53 no 3 pp 955ndash969 2008
Journal of Ecosystems 9
[26] P AasMM Lyons R Pledger D LMitchell andWH JeffreyldquoInhibition of bacterial activities by solar radiation in nearshorewaters and the Gulf of Mexicordquo Aquatic Microbial Ecology vol11 no 3 pp 229ndash238 1996
[27] R Massana J M Gasol P K Bjoslashrnsen et al ldquoMeasurement ofbacterial size via image analysis of epifluorescence preparationsdescription of an inexpensive system and solutions to some ofthe most common problemsrdquo Scientia Marina vol 61 no 3 pp397ndash407 1997
[28] R L J R Kepner Jr and J R Pratt ldquoUse of fluorochromes fordirect enumeration of total bacteria in environmental samplespast and presentrdquo Microbiological Reviews vol 58 no 4 pp603ndash615 1994
[29] J Liu F B Dazzo O Glagoleva B Yu andA K Jain ldquoCMEIASa computer-aided system for the image analysis of bacterialmorphotypes in microbial communitiesrdquo Microbial Ecologyvol 41 no 3 pp 173ndash194 2001
[30] S Norland ldquoThe relationship between biomass and volume ofbacteriardquo inHandbook of Methods in Aquatic Microbial EcologyP F Kemp B F Sherr E B Sherr and J J Cole Eds pp 339ndash345 Lewis Chelsea Mich USA 1993
[31] D C Simon and F Azam ldquoA simple economical method formeasuring bacterial protein synthesis rates in sea water using3H-leucinerdquo Marine Microbial Food Webs vol 6 pp 107ndash1091992
[32] D Kirchman ldquoMeasuring bacterial biomass production andgrowth rates from leucine incorporation in natural aquatic envi-ronmentsrdquo in MarIne MicrobiologymdashMethods In MicrobiologyJ H Paul Ed pp 227ndash237 Academic Press San Diego CalifUSA 30th edition 2001
[33] H L Golterman R S Clymo and M A M OhnstadMethodsfor Physical and Chemical Analysis of Fresh Waters BlackwellPublishing Company London UK 2nd edition 1978
[34] P Del Giorgio ldquoRapid and precise determination of dissolvedoxygen by spectrophotometry evaluation of interference fromcolor and turbidityrdquo Limnology and Oceanography vol 44 no4 pp 1148ndash1154 1999
[35] P A del Giorgio J J Cole and A Cimbleris ldquoRespiration ratesin bacteria exceed phytoplankton production in unproductiveaquatic systemsrdquo Nature vol 385 no 6612 pp 148ndash151 1997
[36] P A del Giorgio and J J Cole ldquoBacterial growth efficiencyin natural aquatic systemsrdquo Annual Review of Ecology andSystematics vol 29 pp 503ndash541 1998
[37] RDevelopmentCoreTeamRALanguage andEnvironment ForStatistical Computing R Foundation for Statistical ComputingVienna Austria 2011 httpwwwR-projectorg
[38] H J de Lange D P Morris and C E Williamson ldquoSolarultraviolet photodegradation of DOCmay stimulate freshwaterfood websrdquo Journal of Plankton Research vol 25 no 1 pp 111ndash117 2003
[39] K Kalinowska ldquoBacteria nanoflagellates and ciliates as com-ponents of the microbial loop in three lakes of different trophicstatusrdquo Polish Journal of Ecology vol 52 no 1 pp 19ndash34 2004
[40] M A Gates ldquoQuantitative importance of ciliates in the plank-tonic biomass of lake ecosystemsrdquoHydrobiologia vol 108 no 3pp 233ndash238 1984
[41] M K Hamdy and O R Noyes ldquoFormation of methyl mercuryby bacteriardquo Journal of Applied Microbiology vol 30 no 3 pp424ndash432 1975
[42] MRavichandran ldquoInteractions betweenmercury and dissolvedorganic mattermdasha reviewrdquo Chemosphere vol 55 no 3 pp 319ndash331 2004
[43] G M Ferrari ldquoInfluence of pH and heavy metals in thedetermination of yellow substance in estuarine areasrdquo RemoteSensing of Environment vol 37 no 2 pp 89ndash100 1991
[44] A M Anesio J Theil-Nielsen and W Graneli ldquoBacterialgrowth on photochemically transformed leachates from aquaticand terrestrial primary producersrdquo Microbial Ecology vol 40no 3 pp 200ndash208 2000
[45] A M Anesio W Graneli G R Aiken D J Kieber andK Mopper ldquoEffect of humic substance photodegradation onbacterial growth and respiration in lake waterrdquo Applied andEnvironmentalMicrobiology vol 71 no 10 pp 6267ndash6275 2005
[46] S P Seitzinger H Hartnett R Lauck et al ldquoMolecular-level chemical characterization and bioavailability of dissolvedorganic matter in stream water using electrospray-ionizationmass spectrometryrdquo Limnology and Oceanography vol 50 no1 pp 1ndash12 2005
[47] J D Wehr J Petersen and S Findlay ldquoInfluence of threecontrasting detrital carbon sources on planktonic bacterialmetabolism in a mesotrophic lakerdquo Microbial Ecology vol 37no 1 pp 23ndash35 1999
[48] O Holm-Hansen E W Helbling B B Prezelin and RC Smith ldquoPolyethylene bags and solar ultraviolet radiationrdquoScience vol 259 no 5094 pp 534ndash535 1993
[49] K A Kuehn M J Lemke K Suberkropp and R G WetzelldquoMicrobial biomass and production associated with decayingleaf litter of the emergent macrophyte Juncus effususrdquo Limnol-ogy and Oceanography vol 45 no 4 pp 862ndash870 2000
[50] E M Gross S Hilt P Lombardo and G Mulderij ldquoSearch-ing for allelopathic effects of submerged macrophytes onphytoplanktonmdashstate of the art and open questionsrdquo Hydrobi-ologia vol 584 no 1 pp 77ndash88 2007
[51] G Mulderij E H van Nes and E van Donk ldquoMacrophyte-phytoplankton interactions the relative importance of allelopa-thy versus other factorsrdquo Ecological Modelling vol 204 no 1-2pp 85ndash92 2007
[52] Q L Wu G Zwart J Wu M P Kamst-van Agterveld S Liuand M W Hahn ldquoSubmersed macrophytes play a key role instructuring bacterioplankton community composition in thelarge shallow subtropical Taihu Lake Chinardquo EnvironmentalMicrobiology vol 9 no 11 pp 2765ndash2774 2007
[53] N HThey D M Marques and R S Souza ldquoLower respirationin the littoral zone of a subtropical shallow lakerdquo Frontiers inMicrobiology vol 3 p 434 2012
1Mean air temperature for the 5 days of incubation2Mean daily flux of GR during incubation (n = 5) 1W = 1 J sminus1 For all other variables n = 33Figures in parentheses are plusmnstandard deviations4119885mean mean depth (m) AT air temperature (∘C) TS total solids (mg Lminus1) Alk alkalinity (120583Eq Lminus1) TN total nitrogen (120583g Lminus1) TP total phosphorus(120583g Lminus1) DOC dissolved organic carbon (mg Lminus1) Abs430 absorbance at 430 nm GR global radiation flux (Wmminus2) LMW low-molecular-weight substances(absorbance at 250 nm)HMW high-molecular-weight substances (absorbance at 365 nm) Abs250 365 (ratio of absorbances at 250 and 365 nm) BP bacterialproduction (120583gC L hminus1) BR bacterial respiration (120583gC L hminus1) BGE bacterial growth efficiency
treatmentfraction suggesting photoinhibition We did notexamine the effect of exposure to light on respiration but ourresults showed no difference in respiration from a previousmeasurement in the field (Table 1) The decrease in BGEwith incubation can be attributed to the increase in respi-ration (even though there was no significant differences inrespiration among fractions it impacted the BGE calculationbecause respiration tended to be higher in bacterial andcontrol fractions)
The general lack of changes in practically all variables inthe bulk fraction indicates that there may be an interactionof grazing with radiation The increase of bacterial densitywith light found in the bacterial fraction may have beenimpeded by grazing in the bulk fraction Higher densitiesof bacteria and bacterial grazers have been found in UV-irradiated DOC suggesting that photo-degradation acts as astimulus to microbial food webs [38] In our case howeverthe decrease in ciliate density with light treatment in thebulk fraction indicated a net effect of photoinhibition onciliates If they did control bacterial density it was at acost of a higher grazing rate per cell On the other side ithas been found that high concentrations of refractory DOClike in humic lakes may be associated to lower bacterialnanoflagellates and ciliates numbers and biomass and hencea less efficient functioning of the microbial loop that islower rates of DOC reincorporation to the food web [39]The density of ciliates in the initial condition (presumably afield concentration estimate) and inside the bags was low (2ndash4 orders of magnitude) when compared to values reportedfor lakes with variable trophic status where they range from19 to 100 times 106 cellsmminus3 (see compilation in Gates [40])Thissuggests that there may be a minor role of ciliates on thedecomposition rates of this refractory DOC in Mangueiralakersquos littoral zones
Unexpectedly we found enhancement in bacterial den-sity biomass and production in the dark treatment of thecontrol fraction This enhancement was first attributed tosome kind of contamination but since no ciliates werefound in these bags and contamination in the three bagssimultaneously is unlikely this result was not disregardedAlthough we were not able to explain this unexpected resultdevelopment of organisms capable of metabolizing mercuryis possible Several common bacterial genera (EscherichiaEnterobacter and Bacillus) are able to resist HgCl
2through
mercury methylation [41]This has compromised the evalua-tion of the photo-degradation alone and needs to be criticallyconsidered
An important point of our study is that we were able tofind significant fading of DOM only in the light treatmentof the bacterial fraction which suggests the existence of acomplementary pathway of dissolved organic degradation bybacteria and light [13] Farjalla et al [11] also found fading(in terms of 250 365 and 430 nm absorbance) of DOM butdecreased bacterial density and production of bacteria inUV-exposed macrophyte leachates indicating that fading itselfsays little about the bioavailability of compounds formedTheincrease in bacterial density together with the stability of theDOC and Abs250 365 suggests that the photodegradationof high-molecular-weight compounds may have producedassimilable substances for bacterial growth [9 10] but theywere readily consumed by bacteria Another important ques-tion still concerning mercury inside control bags is thatthis metal shows great affinity with organic matter alteringthe conformation of the latter [42] other bivalent heavymetals like Cu+2 Pb+2 and Cd+2 have been found to changephotochemistry of yellow substances and humic acids [43]and it is possible that mercury has had some unevaluatednegative effect on photochemical reactivity in the bags whereit was added to
Simultaneous irradiation and incubation in our studymay have exposed bacteria to continuous production ofinhibitory free radicals as suggested by the decrease inbacterial production Many studies that dealt with bacterialutilization of photochemically transformed organic carbonderived from macrophytes employed an approach of pre-treating the leachates with UV light prior to the incubationof bacterial batches [11 13 44] In many cases this priorexposure has been found to inhibit bacterial production anddecrease growth efficiency at least for a short period whichhas been attributed to the formation of hydrogen peroxideand possibly other free radicals by light [11 45] Formationof nonassimilable compounds is also possible Seitzingeret al [46] studied bacterial utilization of dissolved organicmatter in two streams and found that BP increased greatlyduring two days of incubation while DOC concentrationsignificantly decreased (45ndash50) in 12 days However 60of the compounds found in the complex mixture of organicmolecules were not consumed which Seitzinger et al [46]
6 Journal of Ecosystems
Table2Differencesb
etweeninitialcond
ition
andexpo
sition(light)or
not(dark)o
fmacroph
yte-deriv
edDOM
tosunlight
inun
filteredwater
(Bulk)filteredwater
(Bacteria
l)andCon
trol
(HgC
l 2)for120
hun
derinsitucond
ition
sinthesouthern
partof
thesubtropicalshallo
wlake
Mangueira
(sou
thernBrazil)
teste
dby
one-way
ANOVA
andTu
keyrsquos
test(m
eanplusmnsta
ndard
deviation)
Experim
ent
(I)B
ulk
(II)Ba
cterial
(III)C
ontro
lInitial
Light
Dark
Initial
Light
Dark
Initial
Light
Dark
Bacteriald
ensity
(cells10
6mLminus
1 )081plusmn016
a076plusmn050
a051plusmn041
a17
1plusmn031
a265plusmn009
b063plusmn007
c14
3plusmn013
a10
3plusmn010
a218plusmn033
b
Bacterialbiovolume
(120583m
3 )006plusmn002
a005plusmn001
a005plusmn001
a010plusmn001
a008plusmn001
a006plusmn001
a006plusmn001
a007plusmn002
a007plusmn003
a
Bacterialbiomass
(ngC
mLminus
1 )65plusmn026
a372plusmn206
a333plusmn320
a1864plusmn591
a2473plusmn277
a414plusmn045
b95
7plusmn13
1a807plusmn18
5a1649plusmn357
b
Bacterialprodu
ction
(120583gC
Lminus1 hminus1 )
012plusmn004
a028plusmn024
a015plusmn003
a015plusmn001
a002plusmn002
b014plusmn002
a003plusmn001
a003plusmn002
a077plusmn020
b
Bacterialrespiratio
n(120583gC
Lminus1 hminus1 )
NA
NA
109plusmn012
aNA
NA
228plusmn088
aNA
NA
214plusmn12
7a
BacterialG
rowth
Efficiency
NA
NA
011plusmn002
aNA
NA
007plusmn003
aNA
NA
022plusmn012
a
Ciliatedensity
(times10
4cells
mminus3 )
414plusmn18
0a18
0plusmn312
b1560plusmn178a
0plusmn0
0plusmn0
0plusmn0
0plusmn0
0plusmn0
0plusmn0
Diss
olvedOrganicCa
rbon
(mgC
Lminus1 )
1869plusmn087
a1946plusmn112
a2626plusmn78
3a2507plusmn136
a4358plusmn255
a3534plusmn204
a2090plusmn354
a3575plusmn367
a46
10plusmn175a
Abs430
(Abs
430n
m(times10minus2))
251plusmn022
a221plusmn012
a263plusmn019
a269plusmn006
a19
8plusmn014
b235plusmn020
a244plusmn016
ab18
4plusmn035
b271plusmn025
a
Abs250
365
nmratio
085plusmn009
a085plusmn002
a085plusmn004
a076plusmn007
a079plusmn018
a087plusmn002
a075plusmn005
a079plusmn025
a087plusmn006
a
Sign
ificanceo
fone-w
ayANOVA
andTu
keyrsquos
aposte
rioritestatPlt005
isindicatedwith
different
lowercase
superscriptletterswith
ineach
experim
ent(Initialversus
LightversusD
ark)Th
eexceptio
nisbacterial
respira
tionandgrow
theffi
ciencyw
here
differences
weretestedam
ongexperim
ents
NAn
otavailableb
ecause
respira
tionwas
measuredin
dark
bottlesFor
allvariables
n=3replicates
Journal of Ecosystems 7
suggested may have resulted from some inhibition factorinvolved in their utilization Contrastingly Perez et al [12]exposed natural river-water DOM to light also in a simulta-neous exposure and found bacterial enhancement with UVexposure These differences could be due to variations inorigin age and biochemical composition of the DOM [45]
The generally low bioavailability of DOC in the formof humic substances [6] indicates that a large proportionof the dissolved carbon in Lake Mangueira is refractory tobacterial consumption This Abs250 365 ratio is higher inthe littoral zone than in the pelagic zone suggesting higherproportion of low-molecular-weight (LMW Abs 250 nm)substances in the littoral zone when compared to the pelagiczone In fact this was confirmed (pelagic Abs250 mean= 003 standard deviation = 0002 F(14) = 1163 119875 lt0001 littoral zone data obtained from Table 1) In spiteof the smaller size of their molecules these substances canbe less reactive Amon and Benner [4] conducted a cross-environment experiment where they analyzed the reactivityof low-(LMW lt1 KDa) and high-molecular-weight (HMWgt1 KDa) dissolved compounds and found significant changesin BP and BR during the experiment (also five days)Unexpectedly these bacterial variables were higher in HMWsubstrates and based on these results the authors proposedthe size-reactivity continuum model that predicts that themajor path of degradation goes from large highly reactiveto small highly recalcitrant molecules If this hypothesis isbroadly applicable to many ecosystems it suggests that inLakeMangueira the compounds lixiviated by themacrophytebelt had already degraded to some extent and accumulatedas dissolved unreactive LMW compounds with very lowdegradation rates in the littoral zones
The age of the DOM subjected to bacterial degradationcan be fundamental for its availability In our experiment wefocused on the subsequent stage of macrophyte degradationthat is after bacterial and light reworking had alreadyoccurred inside the macrophyte stands Most studies thatinvestigated microbial degradation of macrophytes focusedon the early stages of degradation with exposure of coarsefragments of macrophytes to leaching For example Wehret al [47] found a 45-fold increase in bacterial productionafter 168 h of incubation of macrophyte detritus ConverselyHolm-Hansen et al [48] demonstrated very low rates ofmicrobial degradation of the emergent macrophyte Juncuseffusus only 23of the total biomass in leaf litter bagswas lostafter 268 days Enhancement of bacterial production mightbe related to the early stage of degradation given that Kuehnet al [49] employed senescent leaves in their experiment asituation more similar to ours
One possible explanation for the low photodegradationrates may be the total amount of UV accumulated during theexperiment In the study of Farjalla et al [11] macrophyteleachates were photochemically transformed with total UVenergy asymp5670KJmminus2 which was able to induce a bacterialpositive response This UV intensity is almost 100 timeshigher than that estimated for the field conditions in thepresent study (asymp58KJmminus2) One implication is that underlaboratory conditions the rates of photo-degradation may
be overestimated compared to natural conditions Anotherimportant point however is that polyethylene bags used inthis study may present a reduced rate of UV transmittance(eg 68 of UVB at 300 nm) [48] Even though the trans-mittance increases slightly towards larger wavelengths [26]this quenching may have imposed slightly reduced rates ofphoto-degradation than in the field
We attempted to determine the importance of the twomain routes of carbon degradation in aquatic systemsthat is bacterial and photo-degradation on macrophyte-derived DOM flowing into Lake Mangueira from a largewetland belt Remarkably our results showed few changesin environmental parameters related to carbon degradation(DOC Abs250 365 and respiration) even though bacterialvariables did show changes under lightdark incubationconditions (120 h) This DOM that enters the lake seemsto be highly unreactive in a short-term and may be thecause for the difference in bacterial metabolism between thelittoral and pelagic zones reported by Ng et al [17] sincethis is the main carbon source for bacteria in the littoralzone It is important to take into consideration that it wasthe first approach to the subject in this lake and was aonetime experiment hence more experimental replicationis needed Also there is a need for testing bacterial growthin dilution cultures nutrients amendment effects of light onrespiration and the size and quality spectra of DOM derivedfrom macrophytes and phytoplankton to better address thissubject Another more speculative hypothesis is a possibleallelopathic effect of macrophytes (mainly submersed) sincethis effect on cyanobacteria is largely known [50 51]This hasbeen considered as a possible explanation for lower bacterialdiversity [52] and metabolism [17] in lake zones extensivelycolonized by macrophytes Direct effects on heterotrophicbacteria however still need to be properly addressed
5 Conclusions
In this study we reported a short-term unchange in macro-phyte-derived DOC availability with exposition to sunlightThis suggests that bacterial and photo-degradation can bevery low under ldquoin siturdquo conditions which presents lowerlight intensity when compared to laboratory experimentsbut more evidence is needed to determine if this is apermanent condition of the system This study adds animportant mechanistic explanation (low reactivity) for theprevious finding that bacterial respiration is lower in littoralwhen compared to pelagic zones in lake Mangueira [53] Wehypothesize that given the climatic conditions continuousgrowth of macrophytes and loading of dissolved recalcitrantcompounds contribute to very slow rates ofDOCdegradationand in a short term slight influence of photo-degradationSubmersed and emergent macrophytes cover extensive areasinside and around the drainage channel where we obtainedthe water for the present experiment and similar extensivemacrophyte coverage is common in many lakes across theworld The acknowledgment and further investigation onthis subject are essential for the understanding of carboncycling and metabolism in systems that are densely coveredby macrophytes
8 Journal of Ecosystems
Conflict of Interests
The authors state no conflict of interests involving any step ofthis study
Acknowledgments
The authors draw particular attention to the assistance pro-vided by theChicoMendes Institute of Biodiversity (ICMBio-ESEC TAIM) Also they sincerely thank Dr Laura Utz forhelping with ciliate sampling and handling and Dr LuizKucharski for providing the scintillation counter They thankthe Conselho Nacional de Desenvolvimento Cientıfico eTecnologico of Brazil (CNPq-Long TermEcological ResearchProgram Site 7 Sistema Hidrologico do Taim) for financialsupport They are also grateful to Dr Janet W Reid (JWRAssociates) for revising the English text
References
[1] R G Wetzel ldquoGradient-dominated ecosystems sources andregulatory functions of dissolved organic matter in freshwaterecosystemsrdquo Hydrobiologia vol 229 no 1 pp 181ndash198 1992
[2] G H Lauster P C Hanson and T K Kratz ldquoGross primaryproduction and respiration differences among littoral andpelagic habitats in northernWisconsin lakesrdquoCanadian Journalof Fisheries and Aquatic Sciences vol 63 no 5 pp 1130ndash11412006
[3] K M Docherty K C Young P A Maurice and S DBridgham ldquoDissolved organicmatter concentration and qualityinfluences upon structure and function of freshwater microbialcommunitiesrdquo Microbial Ecology vol 52 no 3 pp 378ndash3882006
[4] R M W Amon and R Benner ldquoBacterial utilization ofdifferent size classes of dissolved organicmatterrdquo Limnology andOceanography vol 41 no 1 pp 41ndash51 1996
[5] L Bracchini A Cozar AM Dattilo et al ldquoThe role of wetlandsin the chromophoric dissolved organic matter release and itsrelation to aquatic ecosystems optical properties A case ofstudy katonga and Bunjako Bays (Victoria Lake Uganda)rdquoChemosphere vol 63 no 7 pp 1170ndash1178 2006
[6] U Munster and R J Chrost ldquoOrigin composition and micro-bial utilization of dissolved organicmatterrdquo inAquaticMicrobialEcology J Overbeck and R J Chrost Eds pp 8ndash46 SpringerNew York NY USA 1990
[7] D O Hessen ldquoDissolved organic carbon in a humic lake effectson bacterial production and respirationrdquo Hydrobiologia vol229 no 1 pp 115ndash123 1992
[8] M J Lindell W Graneli and L J Tranvik ldquoEnhanced bac-terial growth in response to photochemical transformation ofdissolved organicmatterrdquo Limnology andOceanography vol 40no 1 pp 195ndash199 1995
[9] M A Moran and R G Zepp ldquoRole of photoreactions inthe formation of biologically labile compounds from dissolvedorganicmatterrdquo Limnology and Oceanography vol 42 no 6 pp1307ndash1316 1997
[10] S Bertilsson and L J Tranvik ldquoPhotochemically producedcarboxylic acids as substrates for freshwater bacterioplanktonrdquoLimnology and Oceanography vol 43 no 5 pp 885ndash895 1998
[11] V F Farjalla A M Anesio S Bertilsson and W GranelildquoPhotochemical reactivity of aquatic macrophyte leachates abi-otic transformations and bacterial responserdquo Aquatic MicrobialEcology vol 24 no 2 pp 187ndash195 2001
[12] A P Perez M M Diaz M A Ferraro G C Cusminsky andH E Zagarese ldquoReplicated mesocosm study on the role ofnatural ultraviolet radiation in high CDOM shallow lakesrdquoPhotochemical and Photobiological Sciences vol 2 no 2 pp 118ndash123 2003
[13] A M Amado V F Farjalla F D A Esteves R L BozelliF Roland and A Enrich-Prast ldquoComplementary pathwaysof dissolved organic carbon removal pathways in clear-waterAmazonian ecosystems photochemical degradation and bacte-rial uptakerdquo FEMSMicrobiology Ecology vol 56 no 1 pp 8ndash172006
[14] S G Ribblett M A Palmer and D W Coats ldquoThe importanceof bacterivorous protists in the decomposition of stream leaflitterrdquo Freshwater Biology vol 50 no 3 pp 516ndash526 2005
[15] R A Snyder and M P Hoch ldquoConsequences of protist-stimulated bacterial production for estimating protist growthefficienciesrdquo Hydrobiologia vol 341 no 2 pp 113ndash123 1996
[16] N Rooney and J Kalff ldquoInteractions among epilimneticphosphorus phytoplankton biomass and bacterioplanktonmetabolism in lakes of varying submerged macrophyte coverrdquoHydrobiologia vol 501 pp 75ndash81 2003
[17] H-T Ng D da Motta Marques E Jeppesen and MSoslashndergaard ldquoBacterioplankton in the littoral and pelagic zonesof subtropical shallow lakesrdquo Hydrobiologia vol 646 no 1 pp311ndash326 2010
[18] L O Crossetti L S Cardoso V LM Callegaro et al ldquoInfluenceof the hydrological changes on the phytoplankton structureand dynamics in a subtropical wetland-lake systemrdquo ActaLimnologica Brasiliensia vol 19 pp 315ndash329 2007
[19] C R Fragoso Jr D M L M Marques W Collischonn C EM Tucci and E H vanNes ldquoModelling spatial heterogeneity ofphytoplankton in Lake Mangueira a large shallow subtropicallake in South Brazilrdquo Ecological Modelling vol 219 no 1-2 pp125ndash137 2008
[20] J C Ceballos and M L Rodrigues ldquoEstimativa de insolacaomediante satelite geoestacionario resultados preliminaresrdquo inProceedings of 15th Congresso Brasileiro de Meteorologia INPESao Paulo Brazil 2008
[21] R G Wetzel and G E Likens Limnological Analyses SpringerNew York NY USA 3rd edition 2000
[22] American Public Health Association American Water WorksAssociation and Water Environment Federation StandardMethods For the Examination of Water and Wastewater Amer-ican Public Health Associatio Washington DC USA 20thedition 1999
[23] F J H Mackereth J Heron and J F Talling Water AnalysisSome Revised Methods For Limnologists Freshwater BiologicalAssociation Ambleside UK 2nd edition 1989
[24] D J Strome and M C Miller ldquoPhotolytic changes in dissolvedhumic substancesrdquo Verhandlungen der Internationalen Vereini-gung fur Theoretische und Angewandte Limnologie vol 20 pp1248ndash1254 1978
[25] J R Helms A Stubbins J D Ritchie E C Minor D J Kieberand K Mopper ldquoAbsorption spectral slopes and slope ratiosas indicators of molecular weight source and photobleachingof chromophoric dissolved organic matterrdquo Limnology andOceanography vol 53 no 3 pp 955ndash969 2008
Journal of Ecosystems 9
[26] P AasMM Lyons R Pledger D LMitchell andWH JeffreyldquoInhibition of bacterial activities by solar radiation in nearshorewaters and the Gulf of Mexicordquo Aquatic Microbial Ecology vol11 no 3 pp 229ndash238 1996
[27] R Massana J M Gasol P K Bjoslashrnsen et al ldquoMeasurement ofbacterial size via image analysis of epifluorescence preparationsdescription of an inexpensive system and solutions to some ofthe most common problemsrdquo Scientia Marina vol 61 no 3 pp397ndash407 1997
[28] R L J R Kepner Jr and J R Pratt ldquoUse of fluorochromes fordirect enumeration of total bacteria in environmental samplespast and presentrdquo Microbiological Reviews vol 58 no 4 pp603ndash615 1994
[29] J Liu F B Dazzo O Glagoleva B Yu andA K Jain ldquoCMEIASa computer-aided system for the image analysis of bacterialmorphotypes in microbial communitiesrdquo Microbial Ecologyvol 41 no 3 pp 173ndash194 2001
[30] S Norland ldquoThe relationship between biomass and volume ofbacteriardquo inHandbook of Methods in Aquatic Microbial EcologyP F Kemp B F Sherr E B Sherr and J J Cole Eds pp 339ndash345 Lewis Chelsea Mich USA 1993
[31] D C Simon and F Azam ldquoA simple economical method formeasuring bacterial protein synthesis rates in sea water using3H-leucinerdquo Marine Microbial Food Webs vol 6 pp 107ndash1091992
[32] D Kirchman ldquoMeasuring bacterial biomass production andgrowth rates from leucine incorporation in natural aquatic envi-ronmentsrdquo in MarIne MicrobiologymdashMethods In MicrobiologyJ H Paul Ed pp 227ndash237 Academic Press San Diego CalifUSA 30th edition 2001
[33] H L Golterman R S Clymo and M A M OhnstadMethodsfor Physical and Chemical Analysis of Fresh Waters BlackwellPublishing Company London UK 2nd edition 1978
[34] P Del Giorgio ldquoRapid and precise determination of dissolvedoxygen by spectrophotometry evaluation of interference fromcolor and turbidityrdquo Limnology and Oceanography vol 44 no4 pp 1148ndash1154 1999
[35] P A del Giorgio J J Cole and A Cimbleris ldquoRespiration ratesin bacteria exceed phytoplankton production in unproductiveaquatic systemsrdquo Nature vol 385 no 6612 pp 148ndash151 1997
[36] P A del Giorgio and J J Cole ldquoBacterial growth efficiencyin natural aquatic systemsrdquo Annual Review of Ecology andSystematics vol 29 pp 503ndash541 1998
[37] RDevelopmentCoreTeamRALanguage andEnvironment ForStatistical Computing R Foundation for Statistical ComputingVienna Austria 2011 httpwwwR-projectorg
[38] H J de Lange D P Morris and C E Williamson ldquoSolarultraviolet photodegradation of DOCmay stimulate freshwaterfood websrdquo Journal of Plankton Research vol 25 no 1 pp 111ndash117 2003
[39] K Kalinowska ldquoBacteria nanoflagellates and ciliates as com-ponents of the microbial loop in three lakes of different trophicstatusrdquo Polish Journal of Ecology vol 52 no 1 pp 19ndash34 2004
[40] M A Gates ldquoQuantitative importance of ciliates in the plank-tonic biomass of lake ecosystemsrdquoHydrobiologia vol 108 no 3pp 233ndash238 1984
[41] M K Hamdy and O R Noyes ldquoFormation of methyl mercuryby bacteriardquo Journal of Applied Microbiology vol 30 no 3 pp424ndash432 1975
[42] MRavichandran ldquoInteractions betweenmercury and dissolvedorganic mattermdasha reviewrdquo Chemosphere vol 55 no 3 pp 319ndash331 2004
[43] G M Ferrari ldquoInfluence of pH and heavy metals in thedetermination of yellow substance in estuarine areasrdquo RemoteSensing of Environment vol 37 no 2 pp 89ndash100 1991
[44] A M Anesio J Theil-Nielsen and W Graneli ldquoBacterialgrowth on photochemically transformed leachates from aquaticand terrestrial primary producersrdquo Microbial Ecology vol 40no 3 pp 200ndash208 2000
[45] A M Anesio W Graneli G R Aiken D J Kieber andK Mopper ldquoEffect of humic substance photodegradation onbacterial growth and respiration in lake waterrdquo Applied andEnvironmentalMicrobiology vol 71 no 10 pp 6267ndash6275 2005
[46] S P Seitzinger H Hartnett R Lauck et al ldquoMolecular-level chemical characterization and bioavailability of dissolvedorganic matter in stream water using electrospray-ionizationmass spectrometryrdquo Limnology and Oceanography vol 50 no1 pp 1ndash12 2005
[47] J D Wehr J Petersen and S Findlay ldquoInfluence of threecontrasting detrital carbon sources on planktonic bacterialmetabolism in a mesotrophic lakerdquo Microbial Ecology vol 37no 1 pp 23ndash35 1999
[48] O Holm-Hansen E W Helbling B B Prezelin and RC Smith ldquoPolyethylene bags and solar ultraviolet radiationrdquoScience vol 259 no 5094 pp 534ndash535 1993
[49] K A Kuehn M J Lemke K Suberkropp and R G WetzelldquoMicrobial biomass and production associated with decayingleaf litter of the emergent macrophyte Juncus effususrdquo Limnol-ogy and Oceanography vol 45 no 4 pp 862ndash870 2000
[50] E M Gross S Hilt P Lombardo and G Mulderij ldquoSearch-ing for allelopathic effects of submerged macrophytes onphytoplanktonmdashstate of the art and open questionsrdquo Hydrobi-ologia vol 584 no 1 pp 77ndash88 2007
[51] G Mulderij E H van Nes and E van Donk ldquoMacrophyte-phytoplankton interactions the relative importance of allelopa-thy versus other factorsrdquo Ecological Modelling vol 204 no 1-2pp 85ndash92 2007
[52] Q L Wu G Zwart J Wu M P Kamst-van Agterveld S Liuand M W Hahn ldquoSubmersed macrophytes play a key role instructuring bacterioplankton community composition in thelarge shallow subtropical Taihu Lake Chinardquo EnvironmentalMicrobiology vol 9 no 11 pp 2765ndash2774 2007
[53] N HThey D M Marques and R S Souza ldquoLower respirationin the littoral zone of a subtropical shallow lakerdquo Frontiers inMicrobiology vol 3 p 434 2012
suggested may have resulted from some inhibition factorinvolved in their utilization Contrastingly Perez et al [12]exposed natural river-water DOM to light also in a simulta-neous exposure and found bacterial enhancement with UVexposure These differences could be due to variations inorigin age and biochemical composition of the DOM [45]
The generally low bioavailability of DOC in the formof humic substances [6] indicates that a large proportionof the dissolved carbon in Lake Mangueira is refractory tobacterial consumption This Abs250 365 ratio is higher inthe littoral zone than in the pelagic zone suggesting higherproportion of low-molecular-weight (LMW Abs 250 nm)substances in the littoral zone when compared to the pelagiczone In fact this was confirmed (pelagic Abs250 mean= 003 standard deviation = 0002 F(14) = 1163 119875 lt0001 littoral zone data obtained from Table 1) In spiteof the smaller size of their molecules these substances canbe less reactive Amon and Benner [4] conducted a cross-environment experiment where they analyzed the reactivityof low-(LMW lt1 KDa) and high-molecular-weight (HMWgt1 KDa) dissolved compounds and found significant changesin BP and BR during the experiment (also five days)Unexpectedly these bacterial variables were higher in HMWsubstrates and based on these results the authors proposedthe size-reactivity continuum model that predicts that themajor path of degradation goes from large highly reactiveto small highly recalcitrant molecules If this hypothesis isbroadly applicable to many ecosystems it suggests that inLakeMangueira the compounds lixiviated by themacrophytebelt had already degraded to some extent and accumulatedas dissolved unreactive LMW compounds with very lowdegradation rates in the littoral zones
The age of the DOM subjected to bacterial degradationcan be fundamental for its availability In our experiment wefocused on the subsequent stage of macrophyte degradationthat is after bacterial and light reworking had alreadyoccurred inside the macrophyte stands Most studies thatinvestigated microbial degradation of macrophytes focusedon the early stages of degradation with exposure of coarsefragments of macrophytes to leaching For example Wehret al [47] found a 45-fold increase in bacterial productionafter 168 h of incubation of macrophyte detritus ConverselyHolm-Hansen et al [48] demonstrated very low rates ofmicrobial degradation of the emergent macrophyte Juncuseffusus only 23of the total biomass in leaf litter bagswas lostafter 268 days Enhancement of bacterial production mightbe related to the early stage of degradation given that Kuehnet al [49] employed senescent leaves in their experiment asituation more similar to ours
One possible explanation for the low photodegradationrates may be the total amount of UV accumulated during theexperiment In the study of Farjalla et al [11] macrophyteleachates were photochemically transformed with total UVenergy asymp5670KJmminus2 which was able to induce a bacterialpositive response This UV intensity is almost 100 timeshigher than that estimated for the field conditions in thepresent study (asymp58KJmminus2) One implication is that underlaboratory conditions the rates of photo-degradation may
be overestimated compared to natural conditions Anotherimportant point however is that polyethylene bags used inthis study may present a reduced rate of UV transmittance(eg 68 of UVB at 300 nm) [48] Even though the trans-mittance increases slightly towards larger wavelengths [26]this quenching may have imposed slightly reduced rates ofphoto-degradation than in the field
We attempted to determine the importance of the twomain routes of carbon degradation in aquatic systemsthat is bacterial and photo-degradation on macrophyte-derived DOM flowing into Lake Mangueira from a largewetland belt Remarkably our results showed few changesin environmental parameters related to carbon degradation(DOC Abs250 365 and respiration) even though bacterialvariables did show changes under lightdark incubationconditions (120 h) This DOM that enters the lake seemsto be highly unreactive in a short-term and may be thecause for the difference in bacterial metabolism between thelittoral and pelagic zones reported by Ng et al [17] sincethis is the main carbon source for bacteria in the littoralzone It is important to take into consideration that it wasthe first approach to the subject in this lake and was aonetime experiment hence more experimental replicationis needed Also there is a need for testing bacterial growthin dilution cultures nutrients amendment effects of light onrespiration and the size and quality spectra of DOM derivedfrom macrophytes and phytoplankton to better address thissubject Another more speculative hypothesis is a possibleallelopathic effect of macrophytes (mainly submersed) sincethis effect on cyanobacteria is largely known [50 51]This hasbeen considered as a possible explanation for lower bacterialdiversity [52] and metabolism [17] in lake zones extensivelycolonized by macrophytes Direct effects on heterotrophicbacteria however still need to be properly addressed
5 Conclusions
In this study we reported a short-term unchange in macro-phyte-derived DOC availability with exposition to sunlightThis suggests that bacterial and photo-degradation can bevery low under ldquoin siturdquo conditions which presents lowerlight intensity when compared to laboratory experimentsbut more evidence is needed to determine if this is apermanent condition of the system This study adds animportant mechanistic explanation (low reactivity) for theprevious finding that bacterial respiration is lower in littoralwhen compared to pelagic zones in lake Mangueira [53] Wehypothesize that given the climatic conditions continuousgrowth of macrophytes and loading of dissolved recalcitrantcompounds contribute to very slow rates ofDOCdegradationand in a short term slight influence of photo-degradationSubmersed and emergent macrophytes cover extensive areasinside and around the drainage channel where we obtainedthe water for the present experiment and similar extensivemacrophyte coverage is common in many lakes across theworld The acknowledgment and further investigation onthis subject are essential for the understanding of carboncycling and metabolism in systems that are densely coveredby macrophytes
8 Journal of Ecosystems
Conflict of Interests
The authors state no conflict of interests involving any step ofthis study
Acknowledgments
The authors draw particular attention to the assistance pro-vided by theChicoMendes Institute of Biodiversity (ICMBio-ESEC TAIM) Also they sincerely thank Dr Laura Utz forhelping with ciliate sampling and handling and Dr LuizKucharski for providing the scintillation counter They thankthe Conselho Nacional de Desenvolvimento Cientıfico eTecnologico of Brazil (CNPq-Long TermEcological ResearchProgram Site 7 Sistema Hidrologico do Taim) for financialsupport They are also grateful to Dr Janet W Reid (JWRAssociates) for revising the English text
References
[1] R G Wetzel ldquoGradient-dominated ecosystems sources andregulatory functions of dissolved organic matter in freshwaterecosystemsrdquo Hydrobiologia vol 229 no 1 pp 181ndash198 1992
[2] G H Lauster P C Hanson and T K Kratz ldquoGross primaryproduction and respiration differences among littoral andpelagic habitats in northernWisconsin lakesrdquoCanadian Journalof Fisheries and Aquatic Sciences vol 63 no 5 pp 1130ndash11412006
[3] K M Docherty K C Young P A Maurice and S DBridgham ldquoDissolved organicmatter concentration and qualityinfluences upon structure and function of freshwater microbialcommunitiesrdquo Microbial Ecology vol 52 no 3 pp 378ndash3882006
[4] R M W Amon and R Benner ldquoBacterial utilization ofdifferent size classes of dissolved organicmatterrdquo Limnology andOceanography vol 41 no 1 pp 41ndash51 1996
[5] L Bracchini A Cozar AM Dattilo et al ldquoThe role of wetlandsin the chromophoric dissolved organic matter release and itsrelation to aquatic ecosystems optical properties A case ofstudy katonga and Bunjako Bays (Victoria Lake Uganda)rdquoChemosphere vol 63 no 7 pp 1170ndash1178 2006
[6] U Munster and R J Chrost ldquoOrigin composition and micro-bial utilization of dissolved organicmatterrdquo inAquaticMicrobialEcology J Overbeck and R J Chrost Eds pp 8ndash46 SpringerNew York NY USA 1990
[7] D O Hessen ldquoDissolved organic carbon in a humic lake effectson bacterial production and respirationrdquo Hydrobiologia vol229 no 1 pp 115ndash123 1992
[8] M J Lindell W Graneli and L J Tranvik ldquoEnhanced bac-terial growth in response to photochemical transformation ofdissolved organicmatterrdquo Limnology andOceanography vol 40no 1 pp 195ndash199 1995
[9] M A Moran and R G Zepp ldquoRole of photoreactions inthe formation of biologically labile compounds from dissolvedorganicmatterrdquo Limnology and Oceanography vol 42 no 6 pp1307ndash1316 1997
[10] S Bertilsson and L J Tranvik ldquoPhotochemically producedcarboxylic acids as substrates for freshwater bacterioplanktonrdquoLimnology and Oceanography vol 43 no 5 pp 885ndash895 1998
[11] V F Farjalla A M Anesio S Bertilsson and W GranelildquoPhotochemical reactivity of aquatic macrophyte leachates abi-otic transformations and bacterial responserdquo Aquatic MicrobialEcology vol 24 no 2 pp 187ndash195 2001
[12] A P Perez M M Diaz M A Ferraro G C Cusminsky andH E Zagarese ldquoReplicated mesocosm study on the role ofnatural ultraviolet radiation in high CDOM shallow lakesrdquoPhotochemical and Photobiological Sciences vol 2 no 2 pp 118ndash123 2003
[13] A M Amado V F Farjalla F D A Esteves R L BozelliF Roland and A Enrich-Prast ldquoComplementary pathwaysof dissolved organic carbon removal pathways in clear-waterAmazonian ecosystems photochemical degradation and bacte-rial uptakerdquo FEMSMicrobiology Ecology vol 56 no 1 pp 8ndash172006
[14] S G Ribblett M A Palmer and D W Coats ldquoThe importanceof bacterivorous protists in the decomposition of stream leaflitterrdquo Freshwater Biology vol 50 no 3 pp 516ndash526 2005
[15] R A Snyder and M P Hoch ldquoConsequences of protist-stimulated bacterial production for estimating protist growthefficienciesrdquo Hydrobiologia vol 341 no 2 pp 113ndash123 1996
[16] N Rooney and J Kalff ldquoInteractions among epilimneticphosphorus phytoplankton biomass and bacterioplanktonmetabolism in lakes of varying submerged macrophyte coverrdquoHydrobiologia vol 501 pp 75ndash81 2003
[17] H-T Ng D da Motta Marques E Jeppesen and MSoslashndergaard ldquoBacterioplankton in the littoral and pelagic zonesof subtropical shallow lakesrdquo Hydrobiologia vol 646 no 1 pp311ndash326 2010
[18] L O Crossetti L S Cardoso V LM Callegaro et al ldquoInfluenceof the hydrological changes on the phytoplankton structureand dynamics in a subtropical wetland-lake systemrdquo ActaLimnologica Brasiliensia vol 19 pp 315ndash329 2007
[19] C R Fragoso Jr D M L M Marques W Collischonn C EM Tucci and E H vanNes ldquoModelling spatial heterogeneity ofphytoplankton in Lake Mangueira a large shallow subtropicallake in South Brazilrdquo Ecological Modelling vol 219 no 1-2 pp125ndash137 2008
[20] J C Ceballos and M L Rodrigues ldquoEstimativa de insolacaomediante satelite geoestacionario resultados preliminaresrdquo inProceedings of 15th Congresso Brasileiro de Meteorologia INPESao Paulo Brazil 2008
[21] R G Wetzel and G E Likens Limnological Analyses SpringerNew York NY USA 3rd edition 2000
[22] American Public Health Association American Water WorksAssociation and Water Environment Federation StandardMethods For the Examination of Water and Wastewater Amer-ican Public Health Associatio Washington DC USA 20thedition 1999
[23] F J H Mackereth J Heron and J F Talling Water AnalysisSome Revised Methods For Limnologists Freshwater BiologicalAssociation Ambleside UK 2nd edition 1989
[24] D J Strome and M C Miller ldquoPhotolytic changes in dissolvedhumic substancesrdquo Verhandlungen der Internationalen Vereini-gung fur Theoretische und Angewandte Limnologie vol 20 pp1248ndash1254 1978
[25] J R Helms A Stubbins J D Ritchie E C Minor D J Kieberand K Mopper ldquoAbsorption spectral slopes and slope ratiosas indicators of molecular weight source and photobleachingof chromophoric dissolved organic matterrdquo Limnology andOceanography vol 53 no 3 pp 955ndash969 2008
Journal of Ecosystems 9
[26] P AasMM Lyons R Pledger D LMitchell andWH JeffreyldquoInhibition of bacterial activities by solar radiation in nearshorewaters and the Gulf of Mexicordquo Aquatic Microbial Ecology vol11 no 3 pp 229ndash238 1996
[27] R Massana J M Gasol P K Bjoslashrnsen et al ldquoMeasurement ofbacterial size via image analysis of epifluorescence preparationsdescription of an inexpensive system and solutions to some ofthe most common problemsrdquo Scientia Marina vol 61 no 3 pp397ndash407 1997
[28] R L J R Kepner Jr and J R Pratt ldquoUse of fluorochromes fordirect enumeration of total bacteria in environmental samplespast and presentrdquo Microbiological Reviews vol 58 no 4 pp603ndash615 1994
[29] J Liu F B Dazzo O Glagoleva B Yu andA K Jain ldquoCMEIASa computer-aided system for the image analysis of bacterialmorphotypes in microbial communitiesrdquo Microbial Ecologyvol 41 no 3 pp 173ndash194 2001
[30] S Norland ldquoThe relationship between biomass and volume ofbacteriardquo inHandbook of Methods in Aquatic Microbial EcologyP F Kemp B F Sherr E B Sherr and J J Cole Eds pp 339ndash345 Lewis Chelsea Mich USA 1993
[31] D C Simon and F Azam ldquoA simple economical method formeasuring bacterial protein synthesis rates in sea water using3H-leucinerdquo Marine Microbial Food Webs vol 6 pp 107ndash1091992
[32] D Kirchman ldquoMeasuring bacterial biomass production andgrowth rates from leucine incorporation in natural aquatic envi-ronmentsrdquo in MarIne MicrobiologymdashMethods In MicrobiologyJ H Paul Ed pp 227ndash237 Academic Press San Diego CalifUSA 30th edition 2001
[33] H L Golterman R S Clymo and M A M OhnstadMethodsfor Physical and Chemical Analysis of Fresh Waters BlackwellPublishing Company London UK 2nd edition 1978
[34] P Del Giorgio ldquoRapid and precise determination of dissolvedoxygen by spectrophotometry evaluation of interference fromcolor and turbidityrdquo Limnology and Oceanography vol 44 no4 pp 1148ndash1154 1999
[35] P A del Giorgio J J Cole and A Cimbleris ldquoRespiration ratesin bacteria exceed phytoplankton production in unproductiveaquatic systemsrdquo Nature vol 385 no 6612 pp 148ndash151 1997
[36] P A del Giorgio and J J Cole ldquoBacterial growth efficiencyin natural aquatic systemsrdquo Annual Review of Ecology andSystematics vol 29 pp 503ndash541 1998
[37] RDevelopmentCoreTeamRALanguage andEnvironment ForStatistical Computing R Foundation for Statistical ComputingVienna Austria 2011 httpwwwR-projectorg
[38] H J de Lange D P Morris and C E Williamson ldquoSolarultraviolet photodegradation of DOCmay stimulate freshwaterfood websrdquo Journal of Plankton Research vol 25 no 1 pp 111ndash117 2003
[39] K Kalinowska ldquoBacteria nanoflagellates and ciliates as com-ponents of the microbial loop in three lakes of different trophicstatusrdquo Polish Journal of Ecology vol 52 no 1 pp 19ndash34 2004
[40] M A Gates ldquoQuantitative importance of ciliates in the plank-tonic biomass of lake ecosystemsrdquoHydrobiologia vol 108 no 3pp 233ndash238 1984
[41] M K Hamdy and O R Noyes ldquoFormation of methyl mercuryby bacteriardquo Journal of Applied Microbiology vol 30 no 3 pp424ndash432 1975
[42] MRavichandran ldquoInteractions betweenmercury and dissolvedorganic mattermdasha reviewrdquo Chemosphere vol 55 no 3 pp 319ndash331 2004
[43] G M Ferrari ldquoInfluence of pH and heavy metals in thedetermination of yellow substance in estuarine areasrdquo RemoteSensing of Environment vol 37 no 2 pp 89ndash100 1991
[44] A M Anesio J Theil-Nielsen and W Graneli ldquoBacterialgrowth on photochemically transformed leachates from aquaticand terrestrial primary producersrdquo Microbial Ecology vol 40no 3 pp 200ndash208 2000
[45] A M Anesio W Graneli G R Aiken D J Kieber andK Mopper ldquoEffect of humic substance photodegradation onbacterial growth and respiration in lake waterrdquo Applied andEnvironmentalMicrobiology vol 71 no 10 pp 6267ndash6275 2005
[46] S P Seitzinger H Hartnett R Lauck et al ldquoMolecular-level chemical characterization and bioavailability of dissolvedorganic matter in stream water using electrospray-ionizationmass spectrometryrdquo Limnology and Oceanography vol 50 no1 pp 1ndash12 2005
[47] J D Wehr J Petersen and S Findlay ldquoInfluence of threecontrasting detrital carbon sources on planktonic bacterialmetabolism in a mesotrophic lakerdquo Microbial Ecology vol 37no 1 pp 23ndash35 1999
[48] O Holm-Hansen E W Helbling B B Prezelin and RC Smith ldquoPolyethylene bags and solar ultraviolet radiationrdquoScience vol 259 no 5094 pp 534ndash535 1993
[49] K A Kuehn M J Lemke K Suberkropp and R G WetzelldquoMicrobial biomass and production associated with decayingleaf litter of the emergent macrophyte Juncus effususrdquo Limnol-ogy and Oceanography vol 45 no 4 pp 862ndash870 2000
[50] E M Gross S Hilt P Lombardo and G Mulderij ldquoSearch-ing for allelopathic effects of submerged macrophytes onphytoplanktonmdashstate of the art and open questionsrdquo Hydrobi-ologia vol 584 no 1 pp 77ndash88 2007
[51] G Mulderij E H van Nes and E van Donk ldquoMacrophyte-phytoplankton interactions the relative importance of allelopa-thy versus other factorsrdquo Ecological Modelling vol 204 no 1-2pp 85ndash92 2007
[52] Q L Wu G Zwart J Wu M P Kamst-van Agterveld S Liuand M W Hahn ldquoSubmersed macrophytes play a key role instructuring bacterioplankton community composition in thelarge shallow subtropical Taihu Lake Chinardquo EnvironmentalMicrobiology vol 9 no 11 pp 2765ndash2774 2007
[53] N HThey D M Marques and R S Souza ldquoLower respirationin the littoral zone of a subtropical shallow lakerdquo Frontiers inMicrobiology vol 3 p 434 2012
suggested may have resulted from some inhibition factorinvolved in their utilization Contrastingly Perez et al [12]exposed natural river-water DOM to light also in a simulta-neous exposure and found bacterial enhancement with UVexposure These differences could be due to variations inorigin age and biochemical composition of the DOM [45]
The generally low bioavailability of DOC in the formof humic substances [6] indicates that a large proportionof the dissolved carbon in Lake Mangueira is refractory tobacterial consumption This Abs250 365 ratio is higher inthe littoral zone than in the pelagic zone suggesting higherproportion of low-molecular-weight (LMW Abs 250 nm)substances in the littoral zone when compared to the pelagiczone In fact this was confirmed (pelagic Abs250 mean= 003 standard deviation = 0002 F(14) = 1163 119875 lt0001 littoral zone data obtained from Table 1) In spiteof the smaller size of their molecules these substances canbe less reactive Amon and Benner [4] conducted a cross-environment experiment where they analyzed the reactivityof low-(LMW lt1 KDa) and high-molecular-weight (HMWgt1 KDa) dissolved compounds and found significant changesin BP and BR during the experiment (also five days)Unexpectedly these bacterial variables were higher in HMWsubstrates and based on these results the authors proposedthe size-reactivity continuum model that predicts that themajor path of degradation goes from large highly reactiveto small highly recalcitrant molecules If this hypothesis isbroadly applicable to many ecosystems it suggests that inLakeMangueira the compounds lixiviated by themacrophytebelt had already degraded to some extent and accumulatedas dissolved unreactive LMW compounds with very lowdegradation rates in the littoral zones
The age of the DOM subjected to bacterial degradationcan be fundamental for its availability In our experiment wefocused on the subsequent stage of macrophyte degradationthat is after bacterial and light reworking had alreadyoccurred inside the macrophyte stands Most studies thatinvestigated microbial degradation of macrophytes focusedon the early stages of degradation with exposure of coarsefragments of macrophytes to leaching For example Wehret al [47] found a 45-fold increase in bacterial productionafter 168 h of incubation of macrophyte detritus ConverselyHolm-Hansen et al [48] demonstrated very low rates ofmicrobial degradation of the emergent macrophyte Juncuseffusus only 23of the total biomass in leaf litter bagswas lostafter 268 days Enhancement of bacterial production mightbe related to the early stage of degradation given that Kuehnet al [49] employed senescent leaves in their experiment asituation more similar to ours
One possible explanation for the low photodegradationrates may be the total amount of UV accumulated during theexperiment In the study of Farjalla et al [11] macrophyteleachates were photochemically transformed with total UVenergy asymp5670KJmminus2 which was able to induce a bacterialpositive response This UV intensity is almost 100 timeshigher than that estimated for the field conditions in thepresent study (asymp58KJmminus2) One implication is that underlaboratory conditions the rates of photo-degradation may
be overestimated compared to natural conditions Anotherimportant point however is that polyethylene bags used inthis study may present a reduced rate of UV transmittance(eg 68 of UVB at 300 nm) [48] Even though the trans-mittance increases slightly towards larger wavelengths [26]this quenching may have imposed slightly reduced rates ofphoto-degradation than in the field
We attempted to determine the importance of the twomain routes of carbon degradation in aquatic systemsthat is bacterial and photo-degradation on macrophyte-derived DOM flowing into Lake Mangueira from a largewetland belt Remarkably our results showed few changesin environmental parameters related to carbon degradation(DOC Abs250 365 and respiration) even though bacterialvariables did show changes under lightdark incubationconditions (120 h) This DOM that enters the lake seemsto be highly unreactive in a short-term and may be thecause for the difference in bacterial metabolism between thelittoral and pelagic zones reported by Ng et al [17] sincethis is the main carbon source for bacteria in the littoralzone It is important to take into consideration that it wasthe first approach to the subject in this lake and was aonetime experiment hence more experimental replicationis needed Also there is a need for testing bacterial growthin dilution cultures nutrients amendment effects of light onrespiration and the size and quality spectra of DOM derivedfrom macrophytes and phytoplankton to better address thissubject Another more speculative hypothesis is a possibleallelopathic effect of macrophytes (mainly submersed) sincethis effect on cyanobacteria is largely known [50 51]This hasbeen considered as a possible explanation for lower bacterialdiversity [52] and metabolism [17] in lake zones extensivelycolonized by macrophytes Direct effects on heterotrophicbacteria however still need to be properly addressed
5 Conclusions
In this study we reported a short-term unchange in macro-phyte-derived DOC availability with exposition to sunlightThis suggests that bacterial and photo-degradation can bevery low under ldquoin siturdquo conditions which presents lowerlight intensity when compared to laboratory experimentsbut more evidence is needed to determine if this is apermanent condition of the system This study adds animportant mechanistic explanation (low reactivity) for theprevious finding that bacterial respiration is lower in littoralwhen compared to pelagic zones in lake Mangueira [53] Wehypothesize that given the climatic conditions continuousgrowth of macrophytes and loading of dissolved recalcitrantcompounds contribute to very slow rates ofDOCdegradationand in a short term slight influence of photo-degradationSubmersed and emergent macrophytes cover extensive areasinside and around the drainage channel where we obtainedthe water for the present experiment and similar extensivemacrophyte coverage is common in many lakes across theworld The acknowledgment and further investigation onthis subject are essential for the understanding of carboncycling and metabolism in systems that are densely coveredby macrophytes
8 Journal of Ecosystems
Conflict of Interests
The authors state no conflict of interests involving any step ofthis study
Acknowledgments
The authors draw particular attention to the assistance pro-vided by theChicoMendes Institute of Biodiversity (ICMBio-ESEC TAIM) Also they sincerely thank Dr Laura Utz forhelping with ciliate sampling and handling and Dr LuizKucharski for providing the scintillation counter They thankthe Conselho Nacional de Desenvolvimento Cientıfico eTecnologico of Brazil (CNPq-Long TermEcological ResearchProgram Site 7 Sistema Hidrologico do Taim) for financialsupport They are also grateful to Dr Janet W Reid (JWRAssociates) for revising the English text
References
[1] R G Wetzel ldquoGradient-dominated ecosystems sources andregulatory functions of dissolved organic matter in freshwaterecosystemsrdquo Hydrobiologia vol 229 no 1 pp 181ndash198 1992
[2] G H Lauster P C Hanson and T K Kratz ldquoGross primaryproduction and respiration differences among littoral andpelagic habitats in northernWisconsin lakesrdquoCanadian Journalof Fisheries and Aquatic Sciences vol 63 no 5 pp 1130ndash11412006
[3] K M Docherty K C Young P A Maurice and S DBridgham ldquoDissolved organicmatter concentration and qualityinfluences upon structure and function of freshwater microbialcommunitiesrdquo Microbial Ecology vol 52 no 3 pp 378ndash3882006
[4] R M W Amon and R Benner ldquoBacterial utilization ofdifferent size classes of dissolved organicmatterrdquo Limnology andOceanography vol 41 no 1 pp 41ndash51 1996
[5] L Bracchini A Cozar AM Dattilo et al ldquoThe role of wetlandsin the chromophoric dissolved organic matter release and itsrelation to aquatic ecosystems optical properties A case ofstudy katonga and Bunjako Bays (Victoria Lake Uganda)rdquoChemosphere vol 63 no 7 pp 1170ndash1178 2006
[6] U Munster and R J Chrost ldquoOrigin composition and micro-bial utilization of dissolved organicmatterrdquo inAquaticMicrobialEcology J Overbeck and R J Chrost Eds pp 8ndash46 SpringerNew York NY USA 1990
[7] D O Hessen ldquoDissolved organic carbon in a humic lake effectson bacterial production and respirationrdquo Hydrobiologia vol229 no 1 pp 115ndash123 1992
[8] M J Lindell W Graneli and L J Tranvik ldquoEnhanced bac-terial growth in response to photochemical transformation ofdissolved organicmatterrdquo Limnology andOceanography vol 40no 1 pp 195ndash199 1995
[9] M A Moran and R G Zepp ldquoRole of photoreactions inthe formation of biologically labile compounds from dissolvedorganicmatterrdquo Limnology and Oceanography vol 42 no 6 pp1307ndash1316 1997
[10] S Bertilsson and L J Tranvik ldquoPhotochemically producedcarboxylic acids as substrates for freshwater bacterioplanktonrdquoLimnology and Oceanography vol 43 no 5 pp 885ndash895 1998
[11] V F Farjalla A M Anesio S Bertilsson and W GranelildquoPhotochemical reactivity of aquatic macrophyte leachates abi-otic transformations and bacterial responserdquo Aquatic MicrobialEcology vol 24 no 2 pp 187ndash195 2001
[12] A P Perez M M Diaz M A Ferraro G C Cusminsky andH E Zagarese ldquoReplicated mesocosm study on the role ofnatural ultraviolet radiation in high CDOM shallow lakesrdquoPhotochemical and Photobiological Sciences vol 2 no 2 pp 118ndash123 2003
[13] A M Amado V F Farjalla F D A Esteves R L BozelliF Roland and A Enrich-Prast ldquoComplementary pathwaysof dissolved organic carbon removal pathways in clear-waterAmazonian ecosystems photochemical degradation and bacte-rial uptakerdquo FEMSMicrobiology Ecology vol 56 no 1 pp 8ndash172006
[14] S G Ribblett M A Palmer and D W Coats ldquoThe importanceof bacterivorous protists in the decomposition of stream leaflitterrdquo Freshwater Biology vol 50 no 3 pp 516ndash526 2005
[15] R A Snyder and M P Hoch ldquoConsequences of protist-stimulated bacterial production for estimating protist growthefficienciesrdquo Hydrobiologia vol 341 no 2 pp 113ndash123 1996
[16] N Rooney and J Kalff ldquoInteractions among epilimneticphosphorus phytoplankton biomass and bacterioplanktonmetabolism in lakes of varying submerged macrophyte coverrdquoHydrobiologia vol 501 pp 75ndash81 2003
[17] H-T Ng D da Motta Marques E Jeppesen and MSoslashndergaard ldquoBacterioplankton in the littoral and pelagic zonesof subtropical shallow lakesrdquo Hydrobiologia vol 646 no 1 pp311ndash326 2010
[18] L O Crossetti L S Cardoso V LM Callegaro et al ldquoInfluenceof the hydrological changes on the phytoplankton structureand dynamics in a subtropical wetland-lake systemrdquo ActaLimnologica Brasiliensia vol 19 pp 315ndash329 2007
[19] C R Fragoso Jr D M L M Marques W Collischonn C EM Tucci and E H vanNes ldquoModelling spatial heterogeneity ofphytoplankton in Lake Mangueira a large shallow subtropicallake in South Brazilrdquo Ecological Modelling vol 219 no 1-2 pp125ndash137 2008
[20] J C Ceballos and M L Rodrigues ldquoEstimativa de insolacaomediante satelite geoestacionario resultados preliminaresrdquo inProceedings of 15th Congresso Brasileiro de Meteorologia INPESao Paulo Brazil 2008
[21] R G Wetzel and G E Likens Limnological Analyses SpringerNew York NY USA 3rd edition 2000
[22] American Public Health Association American Water WorksAssociation and Water Environment Federation StandardMethods For the Examination of Water and Wastewater Amer-ican Public Health Associatio Washington DC USA 20thedition 1999
[23] F J H Mackereth J Heron and J F Talling Water AnalysisSome Revised Methods For Limnologists Freshwater BiologicalAssociation Ambleside UK 2nd edition 1989
[24] D J Strome and M C Miller ldquoPhotolytic changes in dissolvedhumic substancesrdquo Verhandlungen der Internationalen Vereini-gung fur Theoretische und Angewandte Limnologie vol 20 pp1248ndash1254 1978
[25] J R Helms A Stubbins J D Ritchie E C Minor D J Kieberand K Mopper ldquoAbsorption spectral slopes and slope ratiosas indicators of molecular weight source and photobleachingof chromophoric dissolved organic matterrdquo Limnology andOceanography vol 53 no 3 pp 955ndash969 2008
Journal of Ecosystems 9
[26] P AasMM Lyons R Pledger D LMitchell andWH JeffreyldquoInhibition of bacterial activities by solar radiation in nearshorewaters and the Gulf of Mexicordquo Aquatic Microbial Ecology vol11 no 3 pp 229ndash238 1996
[27] R Massana J M Gasol P K Bjoslashrnsen et al ldquoMeasurement ofbacterial size via image analysis of epifluorescence preparationsdescription of an inexpensive system and solutions to some ofthe most common problemsrdquo Scientia Marina vol 61 no 3 pp397ndash407 1997
[28] R L J R Kepner Jr and J R Pratt ldquoUse of fluorochromes fordirect enumeration of total bacteria in environmental samplespast and presentrdquo Microbiological Reviews vol 58 no 4 pp603ndash615 1994
[29] J Liu F B Dazzo O Glagoleva B Yu andA K Jain ldquoCMEIASa computer-aided system for the image analysis of bacterialmorphotypes in microbial communitiesrdquo Microbial Ecologyvol 41 no 3 pp 173ndash194 2001
[30] S Norland ldquoThe relationship between biomass and volume ofbacteriardquo inHandbook of Methods in Aquatic Microbial EcologyP F Kemp B F Sherr E B Sherr and J J Cole Eds pp 339ndash345 Lewis Chelsea Mich USA 1993
[31] D C Simon and F Azam ldquoA simple economical method formeasuring bacterial protein synthesis rates in sea water using3H-leucinerdquo Marine Microbial Food Webs vol 6 pp 107ndash1091992
[32] D Kirchman ldquoMeasuring bacterial biomass production andgrowth rates from leucine incorporation in natural aquatic envi-ronmentsrdquo in MarIne MicrobiologymdashMethods In MicrobiologyJ H Paul Ed pp 227ndash237 Academic Press San Diego CalifUSA 30th edition 2001
[33] H L Golterman R S Clymo and M A M OhnstadMethodsfor Physical and Chemical Analysis of Fresh Waters BlackwellPublishing Company London UK 2nd edition 1978
[34] P Del Giorgio ldquoRapid and precise determination of dissolvedoxygen by spectrophotometry evaluation of interference fromcolor and turbidityrdquo Limnology and Oceanography vol 44 no4 pp 1148ndash1154 1999
[35] P A del Giorgio J J Cole and A Cimbleris ldquoRespiration ratesin bacteria exceed phytoplankton production in unproductiveaquatic systemsrdquo Nature vol 385 no 6612 pp 148ndash151 1997
[36] P A del Giorgio and J J Cole ldquoBacterial growth efficiencyin natural aquatic systemsrdquo Annual Review of Ecology andSystematics vol 29 pp 503ndash541 1998
[37] RDevelopmentCoreTeamRALanguage andEnvironment ForStatistical Computing R Foundation for Statistical ComputingVienna Austria 2011 httpwwwR-projectorg
[38] H J de Lange D P Morris and C E Williamson ldquoSolarultraviolet photodegradation of DOCmay stimulate freshwaterfood websrdquo Journal of Plankton Research vol 25 no 1 pp 111ndash117 2003
[39] K Kalinowska ldquoBacteria nanoflagellates and ciliates as com-ponents of the microbial loop in three lakes of different trophicstatusrdquo Polish Journal of Ecology vol 52 no 1 pp 19ndash34 2004
[40] M A Gates ldquoQuantitative importance of ciliates in the plank-tonic biomass of lake ecosystemsrdquoHydrobiologia vol 108 no 3pp 233ndash238 1984
[41] M K Hamdy and O R Noyes ldquoFormation of methyl mercuryby bacteriardquo Journal of Applied Microbiology vol 30 no 3 pp424ndash432 1975
[42] MRavichandran ldquoInteractions betweenmercury and dissolvedorganic mattermdasha reviewrdquo Chemosphere vol 55 no 3 pp 319ndash331 2004
[43] G M Ferrari ldquoInfluence of pH and heavy metals in thedetermination of yellow substance in estuarine areasrdquo RemoteSensing of Environment vol 37 no 2 pp 89ndash100 1991
[44] A M Anesio J Theil-Nielsen and W Graneli ldquoBacterialgrowth on photochemically transformed leachates from aquaticand terrestrial primary producersrdquo Microbial Ecology vol 40no 3 pp 200ndash208 2000
[45] A M Anesio W Graneli G R Aiken D J Kieber andK Mopper ldquoEffect of humic substance photodegradation onbacterial growth and respiration in lake waterrdquo Applied andEnvironmentalMicrobiology vol 71 no 10 pp 6267ndash6275 2005
[46] S P Seitzinger H Hartnett R Lauck et al ldquoMolecular-level chemical characterization and bioavailability of dissolvedorganic matter in stream water using electrospray-ionizationmass spectrometryrdquo Limnology and Oceanography vol 50 no1 pp 1ndash12 2005
[47] J D Wehr J Petersen and S Findlay ldquoInfluence of threecontrasting detrital carbon sources on planktonic bacterialmetabolism in a mesotrophic lakerdquo Microbial Ecology vol 37no 1 pp 23ndash35 1999
[48] O Holm-Hansen E W Helbling B B Prezelin and RC Smith ldquoPolyethylene bags and solar ultraviolet radiationrdquoScience vol 259 no 5094 pp 534ndash535 1993
[49] K A Kuehn M J Lemke K Suberkropp and R G WetzelldquoMicrobial biomass and production associated with decayingleaf litter of the emergent macrophyte Juncus effususrdquo Limnol-ogy and Oceanography vol 45 no 4 pp 862ndash870 2000
[50] E M Gross S Hilt P Lombardo and G Mulderij ldquoSearch-ing for allelopathic effects of submerged macrophytes onphytoplanktonmdashstate of the art and open questionsrdquo Hydrobi-ologia vol 584 no 1 pp 77ndash88 2007
[51] G Mulderij E H van Nes and E van Donk ldquoMacrophyte-phytoplankton interactions the relative importance of allelopa-thy versus other factorsrdquo Ecological Modelling vol 204 no 1-2pp 85ndash92 2007
[52] Q L Wu G Zwart J Wu M P Kamst-van Agterveld S Liuand M W Hahn ldquoSubmersed macrophytes play a key role instructuring bacterioplankton community composition in thelarge shallow subtropical Taihu Lake Chinardquo EnvironmentalMicrobiology vol 9 no 11 pp 2765ndash2774 2007
[53] N HThey D M Marques and R S Souza ldquoLower respirationin the littoral zone of a subtropical shallow lakerdquo Frontiers inMicrobiology vol 3 p 434 2012
The authors state no conflict of interests involving any step ofthis study
Acknowledgments
The authors draw particular attention to the assistance pro-vided by theChicoMendes Institute of Biodiversity (ICMBio-ESEC TAIM) Also they sincerely thank Dr Laura Utz forhelping with ciliate sampling and handling and Dr LuizKucharski for providing the scintillation counter They thankthe Conselho Nacional de Desenvolvimento Cientıfico eTecnologico of Brazil (CNPq-Long TermEcological ResearchProgram Site 7 Sistema Hidrologico do Taim) for financialsupport They are also grateful to Dr Janet W Reid (JWRAssociates) for revising the English text
References
[1] R G Wetzel ldquoGradient-dominated ecosystems sources andregulatory functions of dissolved organic matter in freshwaterecosystemsrdquo Hydrobiologia vol 229 no 1 pp 181ndash198 1992
[2] G H Lauster P C Hanson and T K Kratz ldquoGross primaryproduction and respiration differences among littoral andpelagic habitats in northernWisconsin lakesrdquoCanadian Journalof Fisheries and Aquatic Sciences vol 63 no 5 pp 1130ndash11412006
[3] K M Docherty K C Young P A Maurice and S DBridgham ldquoDissolved organicmatter concentration and qualityinfluences upon structure and function of freshwater microbialcommunitiesrdquo Microbial Ecology vol 52 no 3 pp 378ndash3882006
[4] R M W Amon and R Benner ldquoBacterial utilization ofdifferent size classes of dissolved organicmatterrdquo Limnology andOceanography vol 41 no 1 pp 41ndash51 1996
[5] L Bracchini A Cozar AM Dattilo et al ldquoThe role of wetlandsin the chromophoric dissolved organic matter release and itsrelation to aquatic ecosystems optical properties A case ofstudy katonga and Bunjako Bays (Victoria Lake Uganda)rdquoChemosphere vol 63 no 7 pp 1170ndash1178 2006
[6] U Munster and R J Chrost ldquoOrigin composition and micro-bial utilization of dissolved organicmatterrdquo inAquaticMicrobialEcology J Overbeck and R J Chrost Eds pp 8ndash46 SpringerNew York NY USA 1990
[7] D O Hessen ldquoDissolved organic carbon in a humic lake effectson bacterial production and respirationrdquo Hydrobiologia vol229 no 1 pp 115ndash123 1992
[8] M J Lindell W Graneli and L J Tranvik ldquoEnhanced bac-terial growth in response to photochemical transformation ofdissolved organicmatterrdquo Limnology andOceanography vol 40no 1 pp 195ndash199 1995
[9] M A Moran and R G Zepp ldquoRole of photoreactions inthe formation of biologically labile compounds from dissolvedorganicmatterrdquo Limnology and Oceanography vol 42 no 6 pp1307ndash1316 1997
[10] S Bertilsson and L J Tranvik ldquoPhotochemically producedcarboxylic acids as substrates for freshwater bacterioplanktonrdquoLimnology and Oceanography vol 43 no 5 pp 885ndash895 1998
[11] V F Farjalla A M Anesio S Bertilsson and W GranelildquoPhotochemical reactivity of aquatic macrophyte leachates abi-otic transformations and bacterial responserdquo Aquatic MicrobialEcology vol 24 no 2 pp 187ndash195 2001
[12] A P Perez M M Diaz M A Ferraro G C Cusminsky andH E Zagarese ldquoReplicated mesocosm study on the role ofnatural ultraviolet radiation in high CDOM shallow lakesrdquoPhotochemical and Photobiological Sciences vol 2 no 2 pp 118ndash123 2003
[13] A M Amado V F Farjalla F D A Esteves R L BozelliF Roland and A Enrich-Prast ldquoComplementary pathwaysof dissolved organic carbon removal pathways in clear-waterAmazonian ecosystems photochemical degradation and bacte-rial uptakerdquo FEMSMicrobiology Ecology vol 56 no 1 pp 8ndash172006
[14] S G Ribblett M A Palmer and D W Coats ldquoThe importanceof bacterivorous protists in the decomposition of stream leaflitterrdquo Freshwater Biology vol 50 no 3 pp 516ndash526 2005
[15] R A Snyder and M P Hoch ldquoConsequences of protist-stimulated bacterial production for estimating protist growthefficienciesrdquo Hydrobiologia vol 341 no 2 pp 113ndash123 1996
[16] N Rooney and J Kalff ldquoInteractions among epilimneticphosphorus phytoplankton biomass and bacterioplanktonmetabolism in lakes of varying submerged macrophyte coverrdquoHydrobiologia vol 501 pp 75ndash81 2003
[17] H-T Ng D da Motta Marques E Jeppesen and MSoslashndergaard ldquoBacterioplankton in the littoral and pelagic zonesof subtropical shallow lakesrdquo Hydrobiologia vol 646 no 1 pp311ndash326 2010
[18] L O Crossetti L S Cardoso V LM Callegaro et al ldquoInfluenceof the hydrological changes on the phytoplankton structureand dynamics in a subtropical wetland-lake systemrdquo ActaLimnologica Brasiliensia vol 19 pp 315ndash329 2007
[19] C R Fragoso Jr D M L M Marques W Collischonn C EM Tucci and E H vanNes ldquoModelling spatial heterogeneity ofphytoplankton in Lake Mangueira a large shallow subtropicallake in South Brazilrdquo Ecological Modelling vol 219 no 1-2 pp125ndash137 2008
[20] J C Ceballos and M L Rodrigues ldquoEstimativa de insolacaomediante satelite geoestacionario resultados preliminaresrdquo inProceedings of 15th Congresso Brasileiro de Meteorologia INPESao Paulo Brazil 2008
[21] R G Wetzel and G E Likens Limnological Analyses SpringerNew York NY USA 3rd edition 2000
[22] American Public Health Association American Water WorksAssociation and Water Environment Federation StandardMethods For the Examination of Water and Wastewater Amer-ican Public Health Associatio Washington DC USA 20thedition 1999
[23] F J H Mackereth J Heron and J F Talling Water AnalysisSome Revised Methods For Limnologists Freshwater BiologicalAssociation Ambleside UK 2nd edition 1989
[24] D J Strome and M C Miller ldquoPhotolytic changes in dissolvedhumic substancesrdquo Verhandlungen der Internationalen Vereini-gung fur Theoretische und Angewandte Limnologie vol 20 pp1248ndash1254 1978
[25] J R Helms A Stubbins J D Ritchie E C Minor D J Kieberand K Mopper ldquoAbsorption spectral slopes and slope ratiosas indicators of molecular weight source and photobleachingof chromophoric dissolved organic matterrdquo Limnology andOceanography vol 53 no 3 pp 955ndash969 2008
Journal of Ecosystems 9
[26] P AasMM Lyons R Pledger D LMitchell andWH JeffreyldquoInhibition of bacterial activities by solar radiation in nearshorewaters and the Gulf of Mexicordquo Aquatic Microbial Ecology vol11 no 3 pp 229ndash238 1996
[27] R Massana J M Gasol P K Bjoslashrnsen et al ldquoMeasurement ofbacterial size via image analysis of epifluorescence preparationsdescription of an inexpensive system and solutions to some ofthe most common problemsrdquo Scientia Marina vol 61 no 3 pp397ndash407 1997
[28] R L J R Kepner Jr and J R Pratt ldquoUse of fluorochromes fordirect enumeration of total bacteria in environmental samplespast and presentrdquo Microbiological Reviews vol 58 no 4 pp603ndash615 1994
[29] J Liu F B Dazzo O Glagoleva B Yu andA K Jain ldquoCMEIASa computer-aided system for the image analysis of bacterialmorphotypes in microbial communitiesrdquo Microbial Ecologyvol 41 no 3 pp 173ndash194 2001
[30] S Norland ldquoThe relationship between biomass and volume ofbacteriardquo inHandbook of Methods in Aquatic Microbial EcologyP F Kemp B F Sherr E B Sherr and J J Cole Eds pp 339ndash345 Lewis Chelsea Mich USA 1993
[31] D C Simon and F Azam ldquoA simple economical method formeasuring bacterial protein synthesis rates in sea water using3H-leucinerdquo Marine Microbial Food Webs vol 6 pp 107ndash1091992
[32] D Kirchman ldquoMeasuring bacterial biomass production andgrowth rates from leucine incorporation in natural aquatic envi-ronmentsrdquo in MarIne MicrobiologymdashMethods In MicrobiologyJ H Paul Ed pp 227ndash237 Academic Press San Diego CalifUSA 30th edition 2001
[33] H L Golterman R S Clymo and M A M OhnstadMethodsfor Physical and Chemical Analysis of Fresh Waters BlackwellPublishing Company London UK 2nd edition 1978
[34] P Del Giorgio ldquoRapid and precise determination of dissolvedoxygen by spectrophotometry evaluation of interference fromcolor and turbidityrdquo Limnology and Oceanography vol 44 no4 pp 1148ndash1154 1999
[35] P A del Giorgio J J Cole and A Cimbleris ldquoRespiration ratesin bacteria exceed phytoplankton production in unproductiveaquatic systemsrdquo Nature vol 385 no 6612 pp 148ndash151 1997
[36] P A del Giorgio and J J Cole ldquoBacterial growth efficiencyin natural aquatic systemsrdquo Annual Review of Ecology andSystematics vol 29 pp 503ndash541 1998
[37] RDevelopmentCoreTeamRALanguage andEnvironment ForStatistical Computing R Foundation for Statistical ComputingVienna Austria 2011 httpwwwR-projectorg
[38] H J de Lange D P Morris and C E Williamson ldquoSolarultraviolet photodegradation of DOCmay stimulate freshwaterfood websrdquo Journal of Plankton Research vol 25 no 1 pp 111ndash117 2003
[39] K Kalinowska ldquoBacteria nanoflagellates and ciliates as com-ponents of the microbial loop in three lakes of different trophicstatusrdquo Polish Journal of Ecology vol 52 no 1 pp 19ndash34 2004
[40] M A Gates ldquoQuantitative importance of ciliates in the plank-tonic biomass of lake ecosystemsrdquoHydrobiologia vol 108 no 3pp 233ndash238 1984
[41] M K Hamdy and O R Noyes ldquoFormation of methyl mercuryby bacteriardquo Journal of Applied Microbiology vol 30 no 3 pp424ndash432 1975
[42] MRavichandran ldquoInteractions betweenmercury and dissolvedorganic mattermdasha reviewrdquo Chemosphere vol 55 no 3 pp 319ndash331 2004
[43] G M Ferrari ldquoInfluence of pH and heavy metals in thedetermination of yellow substance in estuarine areasrdquo RemoteSensing of Environment vol 37 no 2 pp 89ndash100 1991
[44] A M Anesio J Theil-Nielsen and W Graneli ldquoBacterialgrowth on photochemically transformed leachates from aquaticand terrestrial primary producersrdquo Microbial Ecology vol 40no 3 pp 200ndash208 2000
[45] A M Anesio W Graneli G R Aiken D J Kieber andK Mopper ldquoEffect of humic substance photodegradation onbacterial growth and respiration in lake waterrdquo Applied andEnvironmentalMicrobiology vol 71 no 10 pp 6267ndash6275 2005
[46] S P Seitzinger H Hartnett R Lauck et al ldquoMolecular-level chemical characterization and bioavailability of dissolvedorganic matter in stream water using electrospray-ionizationmass spectrometryrdquo Limnology and Oceanography vol 50 no1 pp 1ndash12 2005
[47] J D Wehr J Petersen and S Findlay ldquoInfluence of threecontrasting detrital carbon sources on planktonic bacterialmetabolism in a mesotrophic lakerdquo Microbial Ecology vol 37no 1 pp 23ndash35 1999
[48] O Holm-Hansen E W Helbling B B Prezelin and RC Smith ldquoPolyethylene bags and solar ultraviolet radiationrdquoScience vol 259 no 5094 pp 534ndash535 1993
[49] K A Kuehn M J Lemke K Suberkropp and R G WetzelldquoMicrobial biomass and production associated with decayingleaf litter of the emergent macrophyte Juncus effususrdquo Limnol-ogy and Oceanography vol 45 no 4 pp 862ndash870 2000
[50] E M Gross S Hilt P Lombardo and G Mulderij ldquoSearch-ing for allelopathic effects of submerged macrophytes onphytoplanktonmdashstate of the art and open questionsrdquo Hydrobi-ologia vol 584 no 1 pp 77ndash88 2007
[51] G Mulderij E H van Nes and E van Donk ldquoMacrophyte-phytoplankton interactions the relative importance of allelopa-thy versus other factorsrdquo Ecological Modelling vol 204 no 1-2pp 85ndash92 2007
[52] Q L Wu G Zwart J Wu M P Kamst-van Agterveld S Liuand M W Hahn ldquoSubmersed macrophytes play a key role instructuring bacterioplankton community composition in thelarge shallow subtropical Taihu Lake Chinardquo EnvironmentalMicrobiology vol 9 no 11 pp 2765ndash2774 2007
[53] N HThey D M Marques and R S Souza ldquoLower respirationin the littoral zone of a subtropical shallow lakerdquo Frontiers inMicrobiology vol 3 p 434 2012
[26] P AasMM Lyons R Pledger D LMitchell andWH JeffreyldquoInhibition of bacterial activities by solar radiation in nearshorewaters and the Gulf of Mexicordquo Aquatic Microbial Ecology vol11 no 3 pp 229ndash238 1996
[27] R Massana J M Gasol P K Bjoslashrnsen et al ldquoMeasurement ofbacterial size via image analysis of epifluorescence preparationsdescription of an inexpensive system and solutions to some ofthe most common problemsrdquo Scientia Marina vol 61 no 3 pp397ndash407 1997
[28] R L J R Kepner Jr and J R Pratt ldquoUse of fluorochromes fordirect enumeration of total bacteria in environmental samplespast and presentrdquo Microbiological Reviews vol 58 no 4 pp603ndash615 1994
[29] J Liu F B Dazzo O Glagoleva B Yu andA K Jain ldquoCMEIASa computer-aided system for the image analysis of bacterialmorphotypes in microbial communitiesrdquo Microbial Ecologyvol 41 no 3 pp 173ndash194 2001
[30] S Norland ldquoThe relationship between biomass and volume ofbacteriardquo inHandbook of Methods in Aquatic Microbial EcologyP F Kemp B F Sherr E B Sherr and J J Cole Eds pp 339ndash345 Lewis Chelsea Mich USA 1993
[31] D C Simon and F Azam ldquoA simple economical method formeasuring bacterial protein synthesis rates in sea water using3H-leucinerdquo Marine Microbial Food Webs vol 6 pp 107ndash1091992
[32] D Kirchman ldquoMeasuring bacterial biomass production andgrowth rates from leucine incorporation in natural aquatic envi-ronmentsrdquo in MarIne MicrobiologymdashMethods In MicrobiologyJ H Paul Ed pp 227ndash237 Academic Press San Diego CalifUSA 30th edition 2001
[33] H L Golterman R S Clymo and M A M OhnstadMethodsfor Physical and Chemical Analysis of Fresh Waters BlackwellPublishing Company London UK 2nd edition 1978
[34] P Del Giorgio ldquoRapid and precise determination of dissolvedoxygen by spectrophotometry evaluation of interference fromcolor and turbidityrdquo Limnology and Oceanography vol 44 no4 pp 1148ndash1154 1999
[35] P A del Giorgio J J Cole and A Cimbleris ldquoRespiration ratesin bacteria exceed phytoplankton production in unproductiveaquatic systemsrdquo Nature vol 385 no 6612 pp 148ndash151 1997
[36] P A del Giorgio and J J Cole ldquoBacterial growth efficiencyin natural aquatic systemsrdquo Annual Review of Ecology andSystematics vol 29 pp 503ndash541 1998
[37] RDevelopmentCoreTeamRALanguage andEnvironment ForStatistical Computing R Foundation for Statistical ComputingVienna Austria 2011 httpwwwR-projectorg
[38] H J de Lange D P Morris and C E Williamson ldquoSolarultraviolet photodegradation of DOCmay stimulate freshwaterfood websrdquo Journal of Plankton Research vol 25 no 1 pp 111ndash117 2003
[39] K Kalinowska ldquoBacteria nanoflagellates and ciliates as com-ponents of the microbial loop in three lakes of different trophicstatusrdquo Polish Journal of Ecology vol 52 no 1 pp 19ndash34 2004
[40] M A Gates ldquoQuantitative importance of ciliates in the plank-tonic biomass of lake ecosystemsrdquoHydrobiologia vol 108 no 3pp 233ndash238 1984
[41] M K Hamdy and O R Noyes ldquoFormation of methyl mercuryby bacteriardquo Journal of Applied Microbiology vol 30 no 3 pp424ndash432 1975
[42] MRavichandran ldquoInteractions betweenmercury and dissolvedorganic mattermdasha reviewrdquo Chemosphere vol 55 no 3 pp 319ndash331 2004
[43] G M Ferrari ldquoInfluence of pH and heavy metals in thedetermination of yellow substance in estuarine areasrdquo RemoteSensing of Environment vol 37 no 2 pp 89ndash100 1991
[44] A M Anesio J Theil-Nielsen and W Graneli ldquoBacterialgrowth on photochemically transformed leachates from aquaticand terrestrial primary producersrdquo Microbial Ecology vol 40no 3 pp 200ndash208 2000
[45] A M Anesio W Graneli G R Aiken D J Kieber andK Mopper ldquoEffect of humic substance photodegradation onbacterial growth and respiration in lake waterrdquo Applied andEnvironmentalMicrobiology vol 71 no 10 pp 6267ndash6275 2005
[46] S P Seitzinger H Hartnett R Lauck et al ldquoMolecular-level chemical characterization and bioavailability of dissolvedorganic matter in stream water using electrospray-ionizationmass spectrometryrdquo Limnology and Oceanography vol 50 no1 pp 1ndash12 2005
[47] J D Wehr J Petersen and S Findlay ldquoInfluence of threecontrasting detrital carbon sources on planktonic bacterialmetabolism in a mesotrophic lakerdquo Microbial Ecology vol 37no 1 pp 23ndash35 1999
[48] O Holm-Hansen E W Helbling B B Prezelin and RC Smith ldquoPolyethylene bags and solar ultraviolet radiationrdquoScience vol 259 no 5094 pp 534ndash535 1993
[49] K A Kuehn M J Lemke K Suberkropp and R G WetzelldquoMicrobial biomass and production associated with decayingleaf litter of the emergent macrophyte Juncus effususrdquo Limnol-ogy and Oceanography vol 45 no 4 pp 862ndash870 2000
[50] E M Gross S Hilt P Lombardo and G Mulderij ldquoSearch-ing for allelopathic effects of submerged macrophytes onphytoplanktonmdashstate of the art and open questionsrdquo Hydrobi-ologia vol 584 no 1 pp 77ndash88 2007
[51] G Mulderij E H van Nes and E van Donk ldquoMacrophyte-phytoplankton interactions the relative importance of allelopa-thy versus other factorsrdquo Ecological Modelling vol 204 no 1-2pp 85ndash92 2007
[52] Q L Wu G Zwart J Wu M P Kamst-van Agterveld S Liuand M W Hahn ldquoSubmersed macrophytes play a key role instructuring bacterioplankton community composition in thelarge shallow subtropical Taihu Lake Chinardquo EnvironmentalMicrobiology vol 9 no 11 pp 2765ndash2774 2007
[53] N HThey D M Marques and R S Souza ldquoLower respirationin the littoral zone of a subtropical shallow lakerdquo Frontiers inMicrobiology vol 3 p 434 2012
