APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1993, p. 3916-3921 Vol. 59, No. 110099-2240/93/113916-06$02.00/0Copyright © 1993, American Society for Microbiology

Bacterioplankton Growth Yield: Seasonal Variations andCoupling to Substrate Lability and

,-Glucosidase ActivitytMATHIAS MIDDELBOE* AND MORTEN S0NDERGAARD

Freshwater Biological Laboratory, University of Copenhagen, Helsing0rsgade 51,DK-3400 Hiller0d, Denmark

Received 2 April 1993/Accepted 25 August 1993

The seasonal variation in the carbon growth yield of pelagic bacteria in the eutrophic lake FrederiksborgSlotss0 was studied. The growth yield was determined in dilution culture experiments, in which a substrate ofdissolved organic carbon (DOC) from the lake was incubated with a natural bacterioplankton assemblage.Bacterial growth efficiency varied annually from 8 to 60%o with an average (and standard deviation) of 41 ±11% (n = 29). Simultaneous measurements of growth yield, substrate lability (DOCL), chlorophyll andbacterial production, abundance, and extracellular enzymatic activity revealed new aspects of the regulation ofbacterial DOC utilization. Growth yield correlated positively to DOCL and negatively to 1-D-glucosidaseactivity. These results indicated a close coupling between the substrate conditions and the physiologicalresponse of the bacteria. The large variations in yield within a few days and the close coupling to substrateavailability showed that one single global carbon yield factor cannot be expected to apply in pelagic systems.

The importance of planktonic bacteria as an energy sourcefor the microbial food web and as mineralizers of carbon andnutrients has been a major research area during the lastdecade (see, e.g., references 12, 37, and 38). The efficiencyof the bacterial transformation of dissolved organic carbon(DOC) into biomass is a key factor in the evaluation of thequantitative role of bacterioplankton in the pelagic food web.

It has long been an accepted approach to use a constant,global value of bacterioplankton carbon growth yield toapply in models and carbon budgets (see, e.g., references 1,2, 9, 12, and 29). The fact that estimates of bacterioplanktongrowth yield during the last decade range from 2 to 80% haspresented the opportunity to argue for almost any conve-nient value to make a budget balance.High growth yields of 50 to 80% have been measured in

short-term experiments of bacterial assimilation of labeledmonosaccharides, amino acids, and microalgal extracellularproducts (4, 23, 45), whereas growth on polymeric leafleachates and on DOC from humic lakes and oligotrophicoceanic waters has resulted in values from 2 to 30% (17, 25,28, 30, 44). In most recent investigations in which the growthyield has been measured from bacteria growing on theambient DOC pool, the values fall within 20 to 40% (7, 8, 26,31, 35, 36).

It has been suggested that part of the observed variationsimply reflects different experimental conditions, e.g., thesource of substrates used, the incubation time, and themethods to measure production, respiration, and/or sub-strate removal (7, 18, 31). If experimental design primarilydetermines the measured growth yield, the bacterial growthyield in a natural environment could still be relativelyconstant.The distribution of the assimilated carbon into biomass

synthesis and respiration and hence the distribution of theenergy derived from respiration between biosynthetic and

* Corresponding author.t Contribution 575 from the Freshwater Biological Laboratory.

maintenance processes ultimately determine the carbongrowth yield of the bacterioplankton.

It has been shown that bacteria can dissociate catabolicand anabolic processes. This means that during periodswhen the growth is limited by the availability of an anabolicsubstrate, ATP may be synthesized in excess of the biosyn-thetic demands (19, 21, 43). Consequently, there is noobligatory coupling between respiration and growth; i.e., thebacterial carbon growth efficiency is theoretically highlyvariable.The aim of this investigation was to determine seasonal

variations in the bacterioplankton growth yield and theinfluence of the substrate conditions on the yield by using thesame procedure throughout the period. The results werecompared with the concentration of labile organic carbonand chlorophyll and the abundance, production, and extra-cellular enzymatic activity of the planktonic bacteria. Thegrowth yield measurements were based on bacterial utiliza-tion of the labile DOC pool in eutrophic lake FrederiksborgSlotss0 during growth in dilution culture experiments. Wehave used direct measurements of carbon in the calculationof net production in order not to rely on conversion factorsfor biovolume estimates and carbon content applied in mostprevious estimations of growth yield.

MATERIALS AND METHODS

Sampling. The study was carried out in FrederiksborgSlotss0 during 1991 and from February to April 1992. Fred-eriksborg Slotss0 is a shallow, eutrophic lake in the northernpart of Zealand, Denmark. It has high bacterial abundance (5x 109 to 35 x 109 cells liter-') and production (up to 250 ,ugof C liter-' day-') (39). The mean annual primary produc-tion is about 400 g of C m-2, and the DOC ranges from 10 to15 mg liter-' and is mainly of autochthonous origin (40).Water was collected at four depths and mixed to represent

an integrated sample of the water column. During stratifica-tion in 1991, sampling included only the epilimnion (depth, 0

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BACTERIOPLANKTON GROWTH YIELD 3917

to 4 m). All chemical and biological measurements werestarted within 1 h after water sampling.

In situ analyses. During spring 1992 a number of in situvariables were determined to monitor the development ofthe phytoplankton biomass and the abundance and activityof the bacterioplankton population.

Bacterial abundance was measured by 4',6-diamidino-2-phenylindole (DAPI) staining (34) and counting of cellsunder epifluorescense microscopy. Bacterial production wasestimated from the incorporation rate of [3H]thymidine intobacterial DNA (14). Thymidine incorporation rates wereconverted to cell production by using a conversion factor of2 x 1018 cells per mol of thymidine incorporated (39).One key feature of bacterial activity is the ability of

bacteria to utilize macromolecules by extracellular enzy-matic activity. Accordingly, the in situ bacterial communitywas physiologically characterized by measurements of theactivity of the extracellular enzyme P3-D-glucosidase. Theenzyme activity was determined as the increase in fluores-cence caused by the enzymatic hydrolysis of the nonfluores-cent substrate 4-methylumbelliferyl-o-D-glucopyranoside,yielding the highly fluorescent 4-methylumbelliferone (11).Only the potential activity at saturated substrate concentra-tions was measured. The samples were incubated at a finalconcentration of 0.5 mM 4-methylumbelliferyl-o-D-glucopy-ranoside for 5 to 7 h. Regular measurements of enzymekinetics and calculation of Vm. ensured that enzyme-sub-strate saturation was attained in the incubations.

Chlorophyll was extracted with 96% ethanol from What-man GF/F filters and measured photometrically as describedpreviously (24).

Decomposition experiments. At each sampling date a de-composition experiment was initiated to estimate the bacte-rioplankton growth yield and the size of the labile fraction ofDOC. Filtered lake water was inoculated with the naturalbacterioplankton assemblage, and the growth was examinedover a 3-week period.The water for media was filtered through precombusted

(550°C for 3 h) Whatman GF/C filters and then subjected tofiltration (pore size, 0.2 p,m) in a tangential flow system(Minitan; Millipore). The inoculum was prepared as a0.8-,um filtrate (Nuclepore) to ensure small initial numbers ofbacteriovores in the cultures. The dilution of the inoculumwas 95%.

Samples for oxygen measurements were placed in gastight50-ml precombusted (550°C for 3 h) bottles. At each sam-pling, two or three bottles were analyzed by the Winklermethod. Samples for measurement of particulate organiccarbon (POC), bacteria, and flagellates were incubated induplicate or triplicate 1,000-ml precombusted Blue Capbottles. Subsamples were analyzed for POC and for bacterialand flagellate abundance. All dilution cultures were incu-bated in the dark at 18°C to avoid any possible influence ofchanges in in situ temperature on the growth yield. Flagellateabundance was measured after DAPI staining as describedfor bacteria above.The utilization of oxygen was converted to CO2 respira-

tion by using a respiratory quotient of 0.82, which representsan annual mean based on a number of determinations ofDOC and oxygen consumption in similar dilution cultures(40). The total DOC consumption during the incubationperiod was termed labile DOC (DOCL) and calculated byadding the amount of respired carbon and the carbon accu-mulated in particles at the end of incubation. A true mea-surement of the DOCL pool requires that growth not belimited by mineral nutrients. In 1992 the medium was

amended with inorganic N and Pi at concentrations of about300 p,g of NH4-N liter-' and 100 ,ug of PO4-P liter, respec-tively.POC was defined as the organic fraction retained on a

GF/F filter. It has been shown that only the smallest bacteria(

3918 MIDDELBOE AND S0NDERGAARD

70

60 4

30-IO0 +

L.. .

0201

10

March April May June July August Sept. Nov. Dec. Jan.

1991FIG. 1. Annual variations in bacterioplankton growth yield in

Frederiksborg Slotss0, 1991. Error bars show the range of themeasurements (n = 2).

occurred on 30 March. The bacterial production had asignificant positive correlation with both the abundance ofbacteria (P < 0.01) and the chlorophyll concentration (P <0.01) (Table 1), whereas bacterial abundance correlatedsignificantly with chlorophyll only if a 4-day delay in thenumerical response was applied (P < 0.02) (Table 1).The concentration of DOCL ranged from 730 to 1,394 ,g

liter-1 (Fig. 3B) and had a significant positive correlationwith growth yield (P < 0.001) and a negative correlation withbacterial abundance (P < 0.06) and bacterial production (P< 0.07) (Table 1).The potential community activity of the extracellular

enzyme ,B-glucosidase ranged from 7.8 to 22.0 nmol liter-'h-1 (data not shown). These measurements were trans-formed to specific activity by being divided by the number ofbacteria (Fig. 3C). The values ranged from 0.37 x 10-9 to1.24 x 10' nmol cell-' h-1 and had a significant negativecorrelation with both bacterial growth yield and the DOCL(P < 0.001) (Table 1).

Contrary to the almost immediate physiological responseof the bacteria to changes in the substrate conditions, asindicated by the close coupling of the extracellular enzy-matic activity and growth yield to changes in DOCL, thevariations in bacterial abundance did not directly covarywith the phytoplankton biomass (Fig. 2; Table 1).

DISCUSSION

The large annual variability in bacterioplankton carbongrowth yield in Frederiksborg Slotss0 (Fig. 1) contradicts thewidespread acceptance of a constant efficiency in bacterialsubstrate utilization. Bacterioplankton growth yield ap-peared to be very dynamic, varying between 10 and 60%within a few days. Consequently, the use of a constant valuein carbon budgets and models is problematic, and we are notyet able to present a simple solution to account for thisvariability.

Determination of growth yield by the dilution cultureapproach is advantageous because it is based on accurate

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1992FIG. 2. Chlorophyll concentrations (A) and bacterial abundance

(-) and production (O) (B) in Frederiksborg Slotss0 in spring 1992.

and direct measurements of production and respiration ofbacteria growing on a natural substrate. Since the sameexperimental procedure underlies all the observed values,the results reflect a true variation in growth yield based onambient DOC.There are, however, some weak points in the procedure.

The substrate composition and concentration were not al-tered during the preparation of the medium, but the dilutionof the bacteria in the cultures improved the specific substratebasis of the inoculated bacteria compared with that under insitu conditions. Consequently, bacterial growth in the cul-tures could be sustained for a relatively longer period beforesubstrate limitation occurred than would have been the casewith an initial bacterial density as in the lake. Typically, theinoculated bacteria reached a stationary growth phase after 3to 5 days of incubation. The growth yield calculation wasbased on the increase in bacterial biomass during the first 3days, which might have resulted in an overestimation of insitu growth yield. The fact that any temperature dependenceof the bacterial growth yield was ignored in the presentinvestigation might have caused deviations from in situgrowth yield. However, Barillier and Gamier (3) found in arecent study that a range of temperature from 8 to 25°C didnot affect bacterioplankton growth yield.The procedure imposes an uncoupling of DOC production

and utilization. Processes such as extracellular release oforganic carbon by phytoplankton, zooplankton grazing, and

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TABLE 1. Correlation coefficients from a multiple regression analysisa

Correlation coefficient" (r) for:Parameter G l-Glucosidase Chlorophyll Bacterial Bacterial abundance

Growth yield DOCL sp act concn abundance (4-day delay in date)

DOCL 0.90***P-Glucosidase sp act -0.96*** -0.94***Chlorophyll concn -0.17 -0.21 0.16 0.72*Bacterial abundance -0.62 -0.62 0.52 0.40Bacterial production -0.53 -0.59 0.53 0.78** 0.76*

a The analysis was calculated by integration between sampling days to allow for changes in rates over time and compensate for some delayed responses.b Levels of significance: ***, P < 0.001; **, P < 0.01; *, P < 0.02.

cell lysis can generate small molecules, which are assimi-lated efficiently and with high affinity and thus are present atlow ambient concentrations (15). How this uncoupling af-fects the growth yield is not known, but the effect is probablyonly slight, since we calculated the growth yield during the

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1992

FIG. 3. Bacterioplankton growth yield (A), DOCL (B), and spe-cific ,B-glucosidase activity (C) in Frederiksborg Slotss0 in spring1992 (mean standard deviation).

initial growth. The procedure we used also prevents miner-alization via grazers. However, any possible effects fromthis were counteracted by the addition of inorganic N and Pi.The high sampling frequency during spring 1992 enabled a

detailed analysis of bacterial substrate dynamics during thisperiod of high biological activity. The inverse relationshipbetween DOCL and the specific ,B-glucosidase activity (Fig.3B and C; Table 1) indicated that the variations in DOCLcovered variations in the pool of monomers and oligomersthat are taken up directly by bacteria without previousextracellular hydrolysis (10). Moreover, the highly signifi-cant correlations between all three variables, DOCL, specific3-glucosidase activity, and bacterioplankton carbon growth

yield (Table 1), suggested that a close coupling existedbetween substrate conditions and the physiological responseof the bacteria.During the initial high DOCL (>1,000 ,ug liter-') from 25

February to 9 March, the specific (3-glucosidase activity wasrelatively low and the bacterial growth yield was 40 to 50%(Fig. 3). Within 3 days after March 9, the DOCL decreased to730 ,ug liter-' (Fig. 3). The negative correlation betweenDOCL and bacterial abundance and production (Table 1)indicated that the decrease in DOCL was caused by bacterialutilization. Assuming a bacterial cell biomass of 22 fg of Ccell-1, the decrease in DOCL during this period was fullyexplained by bacterial production. The decrease in DOCLcorresponded to an increase in 0-glucosidase activity (Fig.3). We therefore suggest that high substrate utilization bybacteria during this period resulted in a depletion of themonomeric and oligomeric compounds. A depletion of easilyavailable substrates would lead to a derepression of 3-glu-cosidase, which might explain the increase in enzymaticactivity (10). We further suggest that the remaining part ofthe labile DOC (>700 ,ug liter-1) consisted mainly of poly-meric compounds and that the increase in potential ,-gluco-sidase activity reflected an actual shift in bacterial substrateutilization toward utilization of polymers.The changes in DOCL were indicative of a short period

with a substantial imbalance in the production and decom-position of DOC. Previous results in Frederiksborg Slotss0showed the balance of DOC production and decompositionover 7-day periods to be within + 15% of the bacterial carbondemand (41). In contrast, the theoretical calculations aboveactually showed the production of bioavailable DOC to beclose to zero during this short period.The negative correlation between enzymatic activity and

growth yield (Table 1) suggests that the energy demandassociated with extracellular hydrolysis leads to a decreasein the efficiency of substrate utilization. Low N and P in thepolymeric substrates might lead to further decreases in theefficiency of the carbon utilization, since the N and P

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3920 MIDDELBOE AND S0NDERGAARD

demand must be sustained by uptake of inorganic N and Pi.The use of inorganic nutrients is energetically more expen-sive than use of organic N and P and leads to morerespiration of organic carbon to meet the increased energydemands (13).

In a similar dilution culture experiment from an oceanicstudy, Kirchman et al. (25) found high turnover rates of DOCwith a 23 to 42% utilization of the total DOC within the first4 days of incubation and with bacterial carbon growth yieldsof 2 to 10%. The nitrogen requirements for biomass produc-tion were supplied mainly by inorganic N, especially asNOf3. The low carbon growth yield might be explained bythe high demand for inorganic N, which indicates highenergy requirements to convert N03-N into organic N.The significant positive correlation between chlorophyll

concentration and bacterial production suggests that phyto-plankton exudates or lysates constituted the main source ofsubstrates for bacterial growth. However, since there was nosignificant correlation between chlorophyll concentrationand 3-glucosidase activity, it was not possible to relatechanges in the physiological response to the increase in thephytoplankton biomass.

Relations between bacterial growth yield and the substrateconditions have previously been discussed in a few cases.Linley and Newell (28) proposed a model describing therelation between the carbon growth yield and the C/N ratioof the bacterial substrate. On the basis of the C/N ratio ofdifferent species of phytoplankton, they calculated a theo-retical range in the carbon growth yield from 38 to 51% forbacteria growing on organic material from these species.Using a similar model, Hopkinson et al. (21) argue that as thesubstrate C/N ratio increases from 6 to 18, the bacteria cansustain a constant growth yield of 33% by decreasing their Nmineralization. At C/N ratios above 18, the growth yielddecreases proportionally to the increase in substrate C/Nratio. A similar relation between substrate C/N ratio andbacterial growth yield was experimentally verified by Gold-man et al. (16), who observed a large increase in growth yieldwhen the C/N ratio of the offered substrate was below 6. Ina study similar to the present one, Barillier and Garnier (3)found a significant positive correlation between growth yieldand DOC and argued that substrate complexity is the mostimportant factor regulating bacterial growth yield.The effects of changing growth conditions on bacterial

growth yield in continuous cultures have been investigatedrecently for natural bacterioplankton assemblages growingon the ambient DOC pool (26, 31). Middelboe et al. (31)found a significant negative correlation between bacterialgrowth yield and population generation time, with growthyields ranging from 20-25% at generation times of about 90 hto 35-45% at generation times of about 20 h. As possibleexplanations they suggested an increase in the relativeimportance of maintenance metabolism and a physiologicalshift of the bacterial population toward utilization of morerefractory substrates at increased generation times. In asimilar study of samples from Frederiksborg Slotss0, Kris-tiansen et al. (26) found the growth yield to vary between 19and 47%, with a tendency to decrease with increasinggeneration time and to decrease with a lowering of mediumquality (from enriched to normal to aged medium). How-ever, none of the correlations were statistically significant.The effect of maintenance metabolism on bacterial growth atincreasing generation times has been experimentally verified(20, 33). If the measured extracellular enzymatic activityreflects actual polymer hydrolysis, the highly significantrelationship between 3-glucosidase activity and growth yield

found in the present investigation suggests that decomposi-tion of polymeric compounds can indeed decrease thegrowth yield. The present results thus support the pervasiveopinion that decomposition of polymeric compounds outsidethe bacterial cell by extracellular enzymes is an importantregulating factor for bacterioplankton production (see, e.g.,references 5, 6, 11, 22, 27, and 32).The correlations presented in this paper strongly suggest

substrate availability as the main regulating factor for bac-terioplankton growth yield in the eutrophic lake Frederiks-borg Slotss0. Moreover, the results provided us with somenew insight into the temporal dynamics of the supply andturnover of DOC during a phytoplankton spring bloom.

ACKNOWLEDGMENTS

This study was supported by the Danish Natural Sciences Re-search Council.

Technical assistance by Gitte Jacobsen is appreciated. Data forbacterial production and abundance in Frederiksborg Slotss0 duringthe spring 1992 were kindly provided by Yvon Letarte. We thankDavid Kirchman and Uwe Munster for valuable comments on themanuscript.

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