rates of microbial degradation of dissolved organic carbon from

Journal of Ptanktoo Research Vol.18 no.9 pp.1521-1533, 1996

Rates of microbial degradation of dissolved organic carbon fromphytoplankton cultures

Wenhao Chen and Peter J.Wangersky1

Department of Oceanography, Dalhousie University, Halifax, Nova Scotia, B3H4JI, Canada

'To whom correspondence should be addressed at: School of Earth and OceanSciences, PO Box 1700, University of Victoria, Victoria, BC, V8W2Y2, Canada

Abstract Dissolved organic carbon (DOC) decay was measured for samples from cultures of thediatoms Chaetoceros grecilii and Phaeodactyhun tricomutum, the flagellate Isochrysis galbana, thedinoflagellate Alexandrium tamarense, and a natural algal assemblage from the Northwest Arm, NovaScotia, Canada, by a high-temperature catalytic oxidation (HTCO) method. Decay rate constants weredetermined using first-order reaction kinetics in the multi-G model of Benter (In Early Diagenais, aTheoretical Approach, Princeton University Press, 1980). Decay rates as high as 037 day-1 wereobtained, which demonstrated that DOC released by phytoplankton might be highly labile to bacterialutilization and could be degraded significantly within hours. Decay rates for most species tested fol-lowed much the same pattern, with KOi values around 03-0.4, A^ values around 0.03, and Km and A^values around 10~3 day1. DOC released by the senescent cells of A.Uonarense was found to be essen-tially bacteria resistant, in contrast to that of the other species tested. The decay of DOC was directlytemperature dependent over the 10-20°C range. Six methods for DOC preservation were tested. Acid-ification with HC1 and refrigerated storage was demonstrated to be the most convenient and practicalmethod. This method can be used for both short- and long-term preservation of DOC samples con-taining highly labile organic compounds.

Introduction

The breakdown of photosynthetically fixed organic carbon in the oceans is one ofthe most important transformations in the global carbon cycle. Yet, there is sur-prisingly little agreement on the processes involved and the rates by which par-ticulate and dissolved organic carbon (POC and DOC) are recycled to carbondioxide and inorganic nutrients. Most DOC in seawater is considered to be resis-tant to microbial mineralization (Barber, 1968; Menzel, 1970). The few estimatesof the fraction that is biologically 'labile' are typically in the range of 1-50% of thetotal DOC (Ammerman et al., 1984; Carlucci et al., 1987). Recent controversy overthe actual quantity of DOC present in seawater suggests that the labile fractioncould have been significantly underestimated, especially in surface waters (Sug-imura and Suzuki, 1988), although the actual size of the underestimation is nowopen to question (Suzuki, 1993).

As observed in studies of POC mineralization (Westrich and Berner, 1984; Pert,1989), bulk seawater DOC is also mineralized in two or more stages (Ogura, 1972,1975).The proportions of these fractions in the total DOC, however, are extremelyvariable in time and space, depending on the activities of the primary producersand their consumers. Consumption and transformation of DOC by bacteriarapidly eliminates much of the low-molecular-weight fraction of DOC, leaving themore refractory materials to accumulate in seawater (Wolter, 1982; Chrost andFaust, 1983; Iturriaga and Zsolnay, 1983; Jensen, 1983; Bell, 1984; Brophy and

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Carlson, 1989). It is indicative of the degree of variability, perhaps locally, in thecomposition of the DOC that other workers have reported faster turnover ratesfor the higher-molecular-weight materials (Araon and Benner, 1994).

Numerous determinations of uptake kinetics for bulk seawater DOC and algalexudates have yielded turnover times ranging from hours to years. Most of thedeterminations were based on either short-term radiotracer investigations ofsimple organic compounds (Wright and Hobbie, 1966; Gocke, 1977; Hagstrom etal., 1984) and phytoplankton exudates and detritus (Iturriaga and Hoppe, 1977;Iturriaga and Zsolnay, 1983; Biddanda, 1988; Pert, 1989), or trace analyses ofextremely labile exudates released by algae (Brockmann et al., 1979; Mopper andLindroth, 1982; Lancelot and Billen, 1984; Bauerfeind, 1985). These studies havegiven some insight into the dynamics of bacterial uptake of simple labile com-ponents of dissolved organic matter in natural waters.

However, these determinations have their limitations.The single substrates addedmight be taken up in a manner different from that of naturally occurring, morecomplex mixtures of solutes. At best, the technique of adding labeled organic com-pounds to samples of seawater measures a rate of utilization of a specific compoundby some small portion of the population present At worst, it selects conditionsfavorable only to bacterial utilization (Wangersky, 1978). On the other hand, nosingle substrate is used with equal efficiency by all bacteria; the choice of substratedetermines the uptake rate measured (Hamilton and Preslan, 1970; Douglas, 1983).Therefore, for all 14C uptake measurements, the question remains as to whether thesimple 14C-labeled compounds used are taken up in the same way as the morevaried and complex 12C substrates already in the water sample (Bauerfeind, 1985).

The uptake rates for the extremely labile compounds do not represent the bac-terial uptake rates for bulk DOC in natural seawater, as demonstrated in thispaper. The kinetic studies on the uptake of simple organic substrates mentionedearlier are normally conducted by the addition of relatively simple compounds oflow molecular weight, such as amino acids and monosaccharides, and are thuslikely to apply only to the relatively small labile component of photoassimilatedcarbon released during the decomposition phase of a phytoplankton bloom.Furthermore, the recent work already mentioned suggests that a portion of theDOC released both during log-phase growth and senescence, unlike the simplepure compounds usually used in addition experiments, is not susceptible to UVphoto-oxidation.The composition of this fraction is unknown, except that it seemsto contain neither nitrogen (Walsh, 1989) nor phosphorus (Ridal and Moore, 1992).

To avoid the problems involved in the above methods, degradation of phyto-plankton detritus has been followed by POC and DOC analyses (Newell et al.,1981) or oxygen consumption (Bauerfeind, 1985). Degradation of intact cells(Fukami et id., 1985) was followed by POC and DOC measurements. However,few rates of DOC degradation have been obtained by these methods. All DOCanalyses in these experiments were done by wet UV or chemical oxidationmethods, which have been shown to underdetennine DOC content significantly(Chen and Wangersky, 1993a,b; Wangersky, 1993). No experiments have been runwith dissolved phytoplankton exudates only, and DOC was not measured by high-temperature catalytic oxidation (HTCO) methods in the experiments reported.

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Rates of degradation of DOC

While a number of researchers have reported little or no difference between wetoxidation and high-temperature combustion analyses of oceanic samples (Oguraand Ogura, 1992; de Baar et al., 1993; Sharp et al., 1993a;Tugrul, 1993), what is actu-ally stated in these papers is that the differences found lie within the variability ofthe two methods. Much of the variability in the HTCO method is the result of uncer-tainty in the method of handling the blank determination, due to the lack of zero-carbon water (Hedges et al., 1993). In our analyses, blank values were determinedusing water which had been subjected to HTCO under conditions more rigorousthan those used in the actual analysis (Chen and Wangersky, 1993a); if any organiccarbon escaped oxidation in the purification process, it would certainly not bedetected in the analysis. Using this water as our blank, we have found amounts oforganic carbon ranging from 25 to 30 uM in water subjected to various treatmentsfor the removal of dissolved organic matter. Since only the machine blank, and notthe carbon contributed by the water 'blank', should be subtracted from the raw data,the use of an improper blank both reduces the amount of DOC found by the HTCOmethod and increases its variability. The precision of our method for DOC is typi-cally in the range of 1-2 uM of carbon, based on a minimum of three replicate injec-tions, with standard and blank samples interspersed throughout the sample runs.The method is discussed in greater detail in Chen and Wangersky (1993b).

Using both HTCO and UV methods, we followed the microbially mediateddecay of DOC in a phytoplankton culture of the diatom Chaetoceros gracilis andin a seawater sample from the Northwest Arm, Nova Scotia, Canada (Chen andWangersky, 1993a). Here, we present further results of our long-term decay experi-ments with DOC from algal cultures of the diatom Phaeodactylum tricomutum, theflagellate Isochrysis galbana, the dinoflagellate Alexandrium tamarense, and anatural algal assemblage from the Northwest Arm, Nova Scotia, Canada.

Recently, there has been an accumulation of evidence showing that bacterialabundance in seawater had been underestimated by an order of magnitude, andthat bacterial production is much higher than was believed in the past (Azam andCho, 1987). Thus, the rate of bacterial utilization of organic matter in the sea mightbe much higher than was thought. High turnover rates of seawater DOC in blooms(0.363 day1; Kirchman et al., 1991) and diatom cultures (0.50 day1; Chen andWangersky, 1993a) have been reported. These results suggest the importance ofsample preservation for DOC measurement; in a region of high primary produc-tivity, the time involved in sample taking and filtration could cause a considerableloss in DOC Without some method of preservation which stops bacterial decom-position of the more biologically labile materials as soon as possible after sampling,we are really determining an undefined portion of the DOC, related in someunknown fashion to the in situ value (Wangersky, 1993). In order to determine aneffective method for the routine preservation of samples, we have tested a numberof sample preservation methods. These results will also be presented here.

Method

The preservation methods for DOC analysis were tested using substrate from aculture of the diatom P.tricornutum in log-phase growth. Aliquots of a 0.8 um

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filtrate of the culture were preserved by six different methods: (i) and (ii) wereboth poisoned by the addition of a 2.5% mercuric chloride solution to a finalconcentration of 0.1 %, acidified by 1 M H Q to a final pH of between 2 and 3, andstored at 2 and 20°C, respectively; (iii) and (iv) were both poisoned with mercuricchloride but not acidified, and stored at 2 and 20°C, respectively; (v) and (vi) wereboth acidified with HC1, and stored at 2 and 20°C, respectively. This test was con-ducted for a relatively short term of 35 days.

Long-term preservation by acidification with HQ and storage in the cold wastested using samples from a bloom culture of the natural phytoplankton popu-lation from the Northwest Arm and seawater from the Bedford Basin, NovaScotia, Canada. About 250 ml of the samples were 0.8 um filtered, acidified withH Q to a pH between 2 and 3, and stored at 2°C in 300 ml glass bottles.

The bottles had previously been combusted at 500°C for 12 h and rinsed withthe filtrates three times to reduce possible sorption by the glass surface (Sharp etal., 1993b). The bottles and samples were sealed tightly with caps with Teflonlinings. Samples for DOC measurements were taken with a 5 ml pipette to avoidany contact between the substrates and the caps. This test was conducted for145 days. In all of the above experiments, samples for DOC analysis were takenat short time intervals at the beginning of the experiment, and at longer intervalslater. DOC was measured by an HTCO method (Chen and Wangersky, 1993a,b).

The organisms used for the decay experiments were grown in batch culture,using the method described elsewhere (Chen and Wangersky, 1993b). When thecultures were brought to the desired growth stage, they were filtered through a 10um Nuclepore filter and then through a 0.8 um Nuclepore filter cartridge bygravity filtration. About 900 ml of filtrate were mixed with -100 ml of 0.8 um fil-tered seawater from the Northwest Arm. This dilution ensured that the naturalbacterial populations in seawater were also present in the experimental samples.For two of the decay samples from the natural algal assemblages, filtered seawa-ter which had been used to leach sediments was also added, to ensure the pres-ence of the natural bacterial population in the samples, and to examine the effectof bacterial species abundance and activity on the turnover of DOC The mixturewas kept in the dark at 20°C and bubbled with organic-free oxygen to prevent itfrom becoming anoxia The bubbling was kept at the lowest rate possible (~3 mlmirr1) to reduce evaporation.

The effect of temperature on the decay rate of DOC was investigated usingculture medium in which the diatom Cgracilis had reached the exponential phaseof growth. Two aliquots of 21 of a 0.8 urn culture filtrate were kept at 10 and 20°Cin the dark. The aliquots were bubbled with 0.22 um filtered compressed air. Theflow rate of the air was ~3 ml min"1. Sampling and analysis were carried out in thesame manner as in the other experiments.

Results and discussion

Sample preservation

The results of short-term sample preservation methods are shown in Table I. Withthe exception of samples acidified with H Q and stored at room temperature, all

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Table L Preservation of DOC samples from a P.tricomutum culture; quantities in micromolar carbon

Day

0137

1435

Hgdj + HCl

20°C

, 128128125129128130

2°C

128128129128126129

HgOj

20°C

128125128127126127

2"C

128126128129125128

HC1

20°C

128126124126123122

2°C

128128125129127127

the methods tested were satisfactory for DOC preservation for at least 35 days.Acidifying with HO alone and storage at room temperature resulted in a consist-ent slow decrease of DOC, with -7% of the DOC lost after 35 days storage. Sinceour analytical precision was of the order of 1-2%, we feel this method of preser-vation for DOC at room temperature, and had been shown previously to be satis-factory for DOC preservation in the cold (2°C) for as long as 87 days (Chen andWangersky, 1993a). Poisoning with mercuric chloride thus seems to be the mostconvenient method for short-term preservation of DOC, and poisoning andstorage in the cold for long-term preservation. However, since mercuric chlorideis a highly toxic substance, it may cause problems in DOC measurement after-wards. It could produce a toxic exhaust gas, harmful to the analyst, or poison thePt catalyst used in the HTCO system (Bauer et al., 1993). No poisoning effect wasobserved in our HTCO system when samples treated with mercuric chloride weremeasured, but the number of samples analysed with this preservative present wasrelatively small.

The results of the long-term preservation studies are shown in Table II. Fluctu-ations around the starting values were within analytical error and exhibited noconsistent trends over the 145 day storage period. DOC samples preserved by thismethod are stable toward bacterial utilization and physical loss. Therefore, of themethods tested, acidification with HC1 and storage in the cold appears to be themost convenient and practical method for DOC preservation for both short andlong periods.

Table IL Preservation of DOC (tiM) from a P.tricomutum culture with HO and storage at 2°C

Day Surface seawater Culture filtrate

~~0 H i 2047 115 212

15 118 20830 113 20562 115 204

100 117 207145 111 203

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Decay experiments

The results of the decay experiments are shown in Figures 1-4 and Table III. Thedata could be described by non-linear equations; however, since the shape of thedecay curves was determined both by decay constants and by amounts of each ofthe fractions of DOC present, particularly of the most labile fraction, the exactequations resulting would depend upon where in the growth and senescence

Table m . DOC (mM) from cultures of A.iamarensc grown in medium originally autoclaved or filtered

Days Autoclaved Filtered

00.090.170.531.0Z04.08.5

15330357.0

287275278288287288295293300292288

273265260265267263270275287283262

C. gradlis

Fig. L Decay of DOC from cultures of Cgracilis.

40 60 80 100 120Days

•*- Senescent -» • Log phase

200

Fig. 2. Decay of DOC from cultures of P.tricomutum.

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240

O;200

160

120

• logPhase • Sene«c«nt

Fig. 3. Decay of DOC from cultures of I.galbana.

20 40 60 80 100 120 140Days

-»- Log phase (added filtrate)

- • - Log phase (no filtrate)

— Senescent (added filtrate)

Fig. 4. Decay of DOC from natural algal assemblage.

process the sample was taken. Comparison between species, or even betweensamples drawn at different times from the same culture, would be difficult or mis-leading. The decay of DOC could also be described by the first-order reactionkinetics of Berner's (1980) multi-G model. In this model, the plot of the log decaycurve is divided into straight-line segments. The slope of each line segment is thedecay rate for the corresponding DOC fraction, and the size of the carbon poolfor that fraction can be calculated.

The decay rates and sizes of pools calculated by the multi-G model are shownin Table IV. The decay rates and pool sizes differed with species, physiologicalstate and culture batch. The rates obtained ranged from 0.12 to 0.49 day"1 for theG01 fraction, 0.02 to 0.08 day1 for the G02 fraction, 7.6 x 10"3 to 0.03 day1 for theG(o fraction, and 1.0 x 10~3to3.7 x 10~3 for the Go4fraction.The pool sizes varied

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Table IV. DOC decay rates

Substrate

CgracUislog phase

CgracUissenescent

P.tricornutumlog phase

P.tricornutumsenescent

I.galbanalog phase

I.galbanasenescent

Natural assemblagelog phase, no additive

Natural assemblagelog phase, added eluate

Natural assemblagesenescent, added eluate

Seawater

Duration (days)

106

88

64

60

34

27

115

107

135

106

DOC pool

Go,Go:Go3Go4

Go,

G02

Go4Go,Go:GmGo4

OO

OO

S 8

S 2

Goi

Go,

Go,G02

G£Go,

GnjGo*

OO

OO

S S

B 2

Go,

%of!DOC

13.519.213.55.8

Hi36.2

9.93.8

10.5337.283

8.916.010.56.8

10.216.0

3.7

10.85.8

13.715.5

6.117.015.114.4

5.015.135.6

20.76.7

K

/Cm

Km

01

^03

Km

Km

Km

Km

Km

KQ\

KQQ

Km,

Rate (day1)

0.480.078.0 X 10-31.0 X 10-3

0.490.080.031.0 X 13-3

0340.020.011.8 x 10-3

0.190.030.01Z7 X 10-3

0.036.5 x 10-3

13 X 10-3

0.120.037.6 x 10-32.9 x 10-3

0370.050.013.1 x lO-3

0310.050.013.7 X 10-3

4.7 x lO-3

4.7 x lO-3

from 5.0% of the total DOC to 135% for Pool GOi, 3.3 to 36.2% for Pool G^, 7.2to 35.6% for Pool G03 and 3.8 to 15.5% for Pool G04

The initial high turnover rates decreased rapidly as the DOC was consumed(Figures l-4,Table IV), decreasing by an order of magnitude within hours to days.In the case of CgracUis, the Goi fraction was gone within a few hours, the G02 frac-tion was present until the third day, the G03 fraction until the 23rd day and the G04fraction was still decaying on the 118th day. After a few weeks of decay, the ratesdecreased to the order of 10"3, two orders of magnitude slower.

The decrease in the rate of bacterial degradation may result from the changingnature of the DOM (Wolter, 1982; Chrost and Faust, 1983; Iturriaga and Zsolnay,1983; Jensen, 1983; Bell, 1984; Brophy and Carlson, 1989). The bacteria consumethe compounds in the order of their ease of oxidation, until only the most resistant

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Rates or degradation of DOC

or nutrient-poor fraction remains. An alternate possibility is that the bacteriarequire an additional nutrient to metabolize DOM, and that this nutrient becomeslimiting as the incubation experiment progresses (Parsons et al., 1980/1981; Kirch-man et al., 1990,1991). In either case, this simple decay experiment has given usinsight into the cycling of DOM in the upper ocean.

The DOC in the cultures of Qgracilis, P.tricomutum and the natural assemblagedecayed rapidly during the first day, with decay rates ranging from 0.12 to 0.49day"1 (see Table IV). The decay rate then slowed down markedly. Most of thelabile DOC was degraded within a few days to a few weeks. After that, the DOCdecayed at a much slower rate. By the end of the experiment, no further decay wasdetectable by our HTCO method. The residual DOC was either resistant to bac-terial degradation or degradable at a rate too slow to be detected over a period ofa few months.

No decay was observed for the DOC from the senescent culture of the dinofla-gellate A.tamarense (Table HI). While some phytoplankton species are known torelease bacteriostatic compounds (Duff et al., 1966; Bruce et al., 1967; Chrost,1975), bacteria are natural constituents of A.tamarense red tides.The lack of decayin this medium suggests that the bacteria normally associated with these red tidessubsist on material coming from sources other than the dinoflagellates. Becauseautoclaving of the medium sometimes results in precipitation of some com-ponents, and thus may affect phytoplankton growth, we ran the experiment withmedium originally sterilized either by autoclaving or by filtration.

The diatom GgracUis, both in log phase and senescence, showed the highestdecay rates and largest pool sizes for the Gm and G02 fractions. This result was con-sistent with the high percentages of low molecular weight observed for DOMfrom this species in both log phase and early senescence, 82 and 84%, respectively(Chen and Wangersky, submitted).

The DOM from the flagellate I.galbana decays much more slowly than that ofthe diatom. Compared to the diatom, in fact, no rate for the GOi pool was observedfor substrate from the culture in mid-log phase, and a rate for the G04 fraction wasobtained only for the substrate from the culture in stationary stage. These resultssuggest that the DOM released by I.galbana is much less labile to bacterial degra-dation, and may possess antibacterial properties (Bruce et al., 1967). This sugges-tion is supported by the result of molecular weight fractionation of the DOMreleased by this species, which was composed largely of high-molecular-weightmaterial (Chen and Wangersky, submitted).

The low values for the decay constants in the seawater sample (see Table IV)suggest that these were in fact K& and K&, the more labile DOM responsible forthe KQI and K^ values having already disappeared, probably before the sampleswere taken.

The substrates from these experimental cultures and from the natural algaeassemblage at different growth stages decayed with significantly different ratesfor the GQI fraction, and with almost identical constants for all other fractions.Thissuggests that the G01 fraction from cultures in log-phase growth is more biolabilethan that in senescent stages. The substrates from senescent cultures of diatomsand the natural algal assemblage, whose bloom species were diatoms (Chen and

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Wangersky, submitted), exhibited high decay rates.The cultures used were in theirearly senescent stage, within 3 days after the population crash, and had high DOCvalues. At that time, a large amount of DOC was released by the senescent cellsas a result of autolysis and decomposition, while bacterial populations had not yethad time to increase. A considerable amount of biolabile DOC still remained inthese cultures. If substrates had been taken from very old cultures, whose mostlabile DOC had already been used by bacteria, the cultures should have displayeddecay constants as low as those observed for seawater in Table IV. It is significantthat the rapid utilization of high-molecular-weight materials observed by Amonand Benner (1994) was with material from a plankton bloom; the smaller mole-cules, which were used much more slowly, may have been composed of com-pounds from the ^03 and KM pools.

The decay rates observed for the DOC from the two batches of the naturalassemblage cultures, both in log phase, differed in KQX, K^ and Km significantly.Thesubstrate with added sediment leaching solution decayed with a KQX three times ashigh, and with K^ and K^ twice as high as those for the substrate without the addedleaching solution. These results suggest that the species, abundance and activity ofbacteria are important factors in determining the turnover rate of the DOC

The decay constants obtained in these experiments demonstrated that biolabileDOC produced by phytoplankton in cultures could be divided into four fractions,decaying in hours to days, days to weeks, weeks to months and months to years,respectively. This is consistent with results obtained by a 14C tracer method forDOC decay from detrital Skletonema costatum cells (Pett, 1989). The amounts ofmaterials falling into each of the fractions were highly variable with species, agesand bacterial activities in the cultures.

The decay rates observed in our experiments might be much higher than naturalor in situ rates since most of the grazers of bacteria (e.g. microflagellates) wereremoved from the decay substrates by 0.8 um filtration. Although the numbers ofbacteria in the decay substrates were not measured, thtlack of grazers should leadto higher than natural seawater bacterial abundances, and hence to increaseddecay rates of DOC. It is obvious that the turnover rates obtained by these experi-ments are specific to the DOM and organisms existing in the substrates used, andcannot be generalized to natural seawater.

The results of the effect of temperature on decay of DOC are shown in Figure5. The bubbling air used in this experiment was not entirely organic free and hencegave some degree of contamination to the decay substrates. The contaminationrate from the compressed air used was monitored using a control sample of SuperQ water with added salt: the DOC increased from 12.5 umol C at the beginning to29.2 umol C by the 60th day. This rate of contamination was relatively minor com-pared to the decay rate of the DOC in the first 30 days. However, it was significantafter that time and became higher than the decay rate of the DOC after -45 days.In spite of the contamination from the bubbling air, the results shown in Figure 5do demonstrate that the decay of DOC by bacteria, and hence the growth of bac-teria, is temperature dependent: it was more rapid at 20°C than at 10°CThis is con-sistent with the work of Takahashi and Ichimura (1971), who found a similartemperature limitation on glucose uptake in seawater.

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30

-»-10C-«-2OC

Fig. 5. Temperature effects on the decay of DOC from a culture of Cgracitis.

Figure 5 shows that the GOi fraction decayed much more slowly at 10°C At 20°C,33.3 umol of DOC disappeared within 0.75 days, with a decay rate of 030 day1,while it took 4.5 days to remove the same amount of DOC at 10°C. Moreover, at10°C, it took two steps to decay this fraction of DOC, with decay rates of 0.15 and0.036 day-1, respectively. After that point, the decay took the same pathway at bothtemperatures. This result suggests that the consumption of highly labile DOC bybacteria is sensitively affected by temperature change.

The high decay rates for the substrates from the diatoms and the natural algalassemblages demonstrated that phytoplankton can release highly labile DOC,which could be utilized by bacteria, and could disappear within hours to days.These results again confirmed the importance of sample processing and preser-vation for DOC measurement in seawater.

Acknowledgements

This work was supported by grants to PJ.W. from the Natural Science and Engin-eering Council of Canada. The loan of instrumentation from the Institute of Bio-marine Sciences of the National Research Council is gratefully acknowledged.

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Received on June 15,1995; accepted on January 26,1996

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