Science of the Total Environment 548–549 (2016) 51–59
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Bacterial production and their role in the removal of dissolved organic matter from tributaries of drinking water reservoirs Norbert Kamjunke a,b,⁎, Marieke R. Oosterwoud c, Peter Herzsprung b, Jörg Tittel b a b c
Helmholtz-Centre for Environmental Research UFZ, Dept. River Ecology, Brückstraße 3a, D-39114 Magdeburg, Germany Helmholtz-Centre for Environmental Research UFZ, Dept. Lake Research, Brückstraße 3a, D-39114 Magdeburg, Germany Helmholtz-Centre for Environmental Research UFZ, Dept. Hydrogeology, Permoserstr. 15, D-04318 Leipzig, Germany
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• DOM was considerably degraded in summer but not during flood events. • The ratio of DOM removal to total DOM release was negatively related to discharge. • Phosphorus addition did not stimulate bacterial DOM degradation. • A proportion of 0.6–12% of the total DOM was degraded in the tributaries.
a r t i c l e
i n f o
Article history: Received 30 October 2015 Received in revised form 4 January 2016 Accepted 4 January 2016 Available online xxxx Editor: D. Barcelo Keywords: Bacterial production DOC Freshness index Humification index Phosphorus
a b s t r a c t Enhanced concentrations of dissolved organic matter (DOM) in freshwaters are an increasing problem in drinking water reservoirs. In this study we investigated bacterial DOM degradation rates in the tributaries of the reservoirs and tested the hypotheses that (1) DOM degradation is high enough to decrease DOM loads to reservoirs considerably, (2) DOM degradation is affected by stream hydrology, and (3) phosphorus addition may stimulate bacterial DOM degradation. Bacterial biomass production, which was used as a measure of DOM degradation, was highest in summer, and was usually lower at upstream than at downstream sites. An important proportion of bacterial production was realized in epilithic biofilms. Production of planktonic and biofilm bacteria was related to water temperature. Planktonic production weakly correlated to DOM quality and to total phosphorus concentration. Addition of soluble reactive phosphorus did not stimulate bacterial DOM degradation. Overall, DOM was considerably degraded in summer at low discharge levels, whereas degradation was negligible during flood events (when DOM load in reservoirs was high). The ratio of DOM degradation to total DOM release was negatively related to discharge. On annual average, only 0.6–12% of total DOM released by the catchments was degraded within the tributaries. © 2016 Elsevier B.V. All rights reserved.
⁎ Corresponding author at: Helmholtz-Centre for Environmental Research UFZ, Dept. River Ecology, Brückstraße 3a, D-39114 Magdeburg, Germany. E-mail address: [email protected] (N. Kamjunke).
http://dx.doi.org/10.1016/j.scitotenv.2016.01.017 0048-9697/© 2016 Elsevier B.V. All rights reserved.
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1. Introduction Globally, streams and lakes receive an estimated amount of 2.0–2.7 billion tons of terrestrial carbon per year (Cole et al., 2007; Battin et al., 2008; Aufdenkampe et al., 2011). The majority of the terrestrial organic carbon entering freshwater systems is respired to CO2 locally or buried in sediments, whereas only a fraction is discharged into the ocean (Aufdenkampe et al., 2011). The organic carbon in rivers and streams affects aquatic ecosystems and serves as a major energy source for microbes (Findlay et al., 1993; Jonsson et al., 2007). Particularly freshly leached, terrestrial dissolved organic matter (DOM) may influence stream ecosystem processes (Burrows et al., 2013). In addition, the dynamics of DOM mobilization is typically linked to hydrology. About 50% of the DOM was exported by the 10% highest discharges (Hilton et al., 1997). Snowmelt events and heavy rainfall events in summer causing high discharges mobilized the highest DOM fluxes in a small catchment, and spring areas with high DOM pools and pronounced hydrological dynamics were regarded as important sources (Andrews et al., 2011). The concept of hot spots and hot moments (e.g., McClain et al., 2003) may be applied to the DOM export from catchments: Pools of available DOM (e.g., upland areas) are hot spots only during certain periods (hot moments; e.g., heavy rainfall) when hydrological connectivity between the terrestrial sources of DOM and stream discharge is facilitated, enabling a relevant mass flux to surface waters (Casper et al., 2003; Stieglitz et al., 2003). Once the DOM has entered the surface water ecosystem, instream heterotrophic processing of terrestrial derived carbon occurs along the river continuum gradient from the headwater downstream (River Continuum Concept, Vannote et al., 1980). DOM is mainly consumed by heterotrophic bacteria, whereby it is partly removed by respiration and partly transferred to bacterial biomass, the latter process making the carbon available to organisms of higher trophic levels. Terrigenous DOM was shown to be respired by microorganisms rather than incorporated into their biomass (Fashing et al., 2014). Besides bacteria suspended in water, bacteria also grow in biofilms which are substratum-associated consortia of microorganisms including microalgae, bacteria, fungi, protozoans and small metazoans, as well as their extracellular polymeric substances (EPS). Biofilms, e.g. on stones, are regarded as major sites of carbon cycling in streams (Romani et al., 2004; Battin et al., 2008). The use of DOM as a bacterial energy source is controlled by the concentration of limiting nutrients (N and/or P), temperature, and the chemical composition of DOM (Stelzer et al., 2003; Lane et al., 2013). Nutrient enrichment increased biofilm development and reduced the bacterial use of autochthonous carbon within the biofilm (Ziegler and Lyon, 2010). Carbon uptake of biofilms was sensitive to temperature (Baldwin et al., 2014). Land use and vegetation type have a major impact on the composition of DOM, affecting the degree of humification within catchments. DOM in agricultural streams was shown to be more labile and thus more accessible for microbes than DOM in wetland streams (Williams et al., 2010). A positive relationship was found between the aromatic content of DOM and the proportion of forested area in the river network of the Bode catchment in Germany (Kamjunke et al., 2013). Regarding the relationship between DOM quality and bacterial processing, planktonic bacterial production was positively related to labile DOM concentration in streams in Southern Ontario (Williams et al., 2010), and microbial bioavailability of DOM was negatively related to the proportions of humic-like DOM in streams of Maryland (Hosen et al., 2014). Recently, Kamjunke et al. (2015) compared planktonic and biofilm bacterial production with patterns of DOC along a topographic and land-use gradient at 17 sampling sites on one occasion (base flow in late summer). It was found that planktonic production was weakly correlated to the total DOC concentration but strongly to DOM quality. Biofilm production, on the other hand, was independent of both DOC concentration and DOM quality. However, little is known about the effect of stream hydrology on bacterial activity. Bacterial production, measured only during
one investigated period, was found to be low at high discharge in Danube floodplains (Peduzzi et al., 2008). Increasing concentrations of dissolved organic matter in freshwaters due to rising input of organic carbon is a common phenomenon in many regions (Hejzlar et al., 2003; Freeman et al., 2004; Eimers et al., 2008). Monteith et al. (2007) observed such an increase in more than 70% of the investigated surface waters in Northern Europe, Great Britain, and North America. An increase in DOM in English streams of 65% between 1988 and 2000 was found to be particularly caused by DOM from the catchment area, as indicated by the enrichment of humic substances (Freeman et al., 2001); the enhancement was even threefold in the Hudson River (Findlay, 2005). In Germany, a significant increase in DOM concentration was observed in 55% of 86 streams (Sucker et al., 2011). The browning of surface waters leads to increasing problems regarding the production of drinking water. Increasing DOC chlorination cause the formation of mutagenic (Nieuwenhuijsen et al., 2000) and carcinogenic (Sadiq and Rodriguez, 2004) disinfection by-products, taste and odor problems as well as growth of potential pathogens, adversely affect the function of water treatment plants and increase costs for purification of drinking water (Ledesma et al., 2012). The amount of chemicals for flocculation and precipitation rises, the running time of filters decreases, and the amount of sludge in water works increases. The corresponding challenge is to find a cost-effective DOM removal technique which makes use of the instream bacterial production potential at different seasons and along the river-continuum gradient with nutrient addition as a possible management practice. In light of these growing concerns regarding increased DOM inputs into aquatic ecosystems, it is crucial that we understand how much instream bacterial production can contribute to the removal of DOM along a downstream gradient at different seasons and flow conditions. Furthermore, there is a need to explore the feasibility of using nutrient additions (P) as a method of stimulating DOM removal. While one traditional aim of reservoir management is to limit phosphorus imports (Cooke et al., 2005) the mineralization of DOC by bacteria can be limited by phosphorus in particular at high DOC to P ratios (Vadstein and Olsen, 1989). The main goal of the present study is to investigate whether the DOM released from the catchments is transported mainly unchanged by the tributaries to the reservoirs, or if the DOM is degraded considerably within the inflow streams, leaving only a part of the released DOM for the load of the reservoir. If the latter were true, one might consider measures of stimulating DOM degradation to reduce the export to the reservoir. Therefore, we tested the following hypotheses that (1) bacterial DOM degradation rates in the tributaries of the reservoir are high enough to decrease DOM concentrations considerably, (2) DOM degradation is affected by stream hydrology, and (3) phosphate addition may release bacteria from phosphorus limitation and thereby stimulate bacterial DOM degradation. 2. Material and methods 2.1. Study sites and sampling Three streams with different properties were selected: Hassel and Rappbode are tributaries leading to the Rappbode reservoir in the Harz Mountains (Germany; see Rinke et al., 2013; Friese et al., 2014 and Tittel et al., 2015 for detailed description), and Rote Mulde in the Western Ore Mountains is a tributary to the Muldenberg reservoir (see Tittel et al., 2013 for further information; Fig. 1). The Rote Mulde was subjected to a long-term increase in DOC concentration between 1995 and 2010 (Tittel et al., 2013). The drainage area of each catchment/sampling site was determined by delineating the subcatchments on a digital elevation model DEM of 4 m × 4 m resolution using the sampling sites as outlets (Whitebox, QGIS). Topographical maps (Atkis, Landesvermessung und Geobasisinformation Sachsen Anhalt) with a resolution of 1:250,000 were used to determine the contribution of the four land-use classes (agricultural, grassland, forest, urban)
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Fig. 1. Map of the two sampling regions: Muldenberg reservoir with the tributary Rote Mulde, and Rappbode reservoir with the tributaries Rappbode and Hassel. Green: forest, yellow: agricultural area, orange: urban area, red dots: sampling sites.
identified within the catchments. The three catchments differ in land use: 98% forest in the Rote Mulde catchment, some agriculture and 72% forest in the Rappbode catchment, and a dominance of agricultural and urban area with only 37% forest in the Hassel catchment (Fig. 1). As a consequence, different DOC and phosphorus patterns were observed: high DOC/low P in Rote Mulde, low DOC/low P in Rappbode, and low DOC/high P in Hassel (Table 1). Sampling was performed between April and October in 2013 and between March and October in 2014. Hassel and Rappbode were sampled six times a year at an upstream and a downstream site each, whereas Rote Mulde (total length 4 km) was sampled only three times a year and only at the catchments outflow before the reservoir. 2.2. Basic parameters Discharge was measured using a conservative tracer. Depending on discharge, between 200 g and 5000 g of table salt (NaCl) was dissolved in stream water in a bucket and added upstream of the sampling site. Conductivity was measured with multiparameter probes (2013: Hydrolab DSX5, OTT Hydromet; 2014: EXO2, Xylem Inc.), and the area under the conductivity curve was used for discharge calculation. The probes also provided data for water temperature and pH. Water depth was measured for three to seven orthogonal transects within the stream for each site. Total phosphorous (TP) was measured using the ammonium molybdate spectrometric method (see Kamjunke et al., 2013). 2.3. DOC analysis For analysis of dissolved organic carbon (DOC), water samples were transferred into acid-rinsed and combusted brown glass bottles, kept at 4 °C for maximum of 24 h and filtered through glass fiber filters
Table 1 Properties of catchments and streams; chemical values represent ranges during samplings in 2013 and 2014. Stream Catchment area (km2) Mean discharge (L s−1) Total phosphorus (TP, μg L−1) DOC (mg L−1)
Rappbode
Hassel
Rote Mulde
39.1 730 15–40 3.2–5.3
40.5 650 14–86 4.0–9.2
5.4 120 7–28 5.6–28.3
(Whatman GF/F). DOC concentrations were determined after hightemperature combustion (DIMATOC 2000, Dimatec Analysentechnik GmbH, Essen, Germany). Fluorescence excitation emission matrices (EEMs) were collected using a spectrofluorometer (AQUALOG, HORIBA Jobin Yvon, USA). Fluorescence intensity was measured during emission scans (240 nm–600 nm every 3.27 nm; 8 pixel) at set excitation wavelengths in 3 nm increments from 240 nm to 600 nm. A 5 nm bandpass for excitation and emission wavelength and 1 s integration time were used. Fluorescence data were corrected (including blank subtraction) using HORIBA internal software before the correction of inner-filter effects (Kothalawa et al., 2013), a method used in many studies. Afterwards, the EEMF spectra were Rayleigh-scattering masked and corrected for inner-filter effects using an AQUALOG internal recorded UV–Vis absorbance spectrum in the same quartz cell of the fluorescent sample. Two fluorescent indices were used to describe the general DOM structure: freshness index and humification index. The freshness index (ß/α, where ß represents more recently derived DOM and α represents highly decomposed DOM) (Fellman et al., 2010) was computed as the ratio of the emission intensity at 380 nm divided by the highest detected emission intensity between 420 and 435 nm, all obtained for excitation at 310 nm. The ratio of ß/α is an indicator of autochthonous inputs and when applied to our data provides an indicator of relative contribution of recent, microbially produced DOM. The humification index HIX was calculated by dividing the peak area under the emission spectra between 435 and 480 nm with the peak area under the emission spectra between 300 and 345 nm, both at 255 nm excitation wavelength (Zsolnay, 2003). The humification index is associated with the condensing of fluorescence molecules and lower H/C ratios and is therefore an indicator of humicity. 2.4. Bacterial DOC utilization Production of stream water bacteria was measured using the leucine technique (Simon and Azam, 1989) as described by Kamjunke et al. (2015). After storage of samples at 4 °C until the next day, triplicate 5 ml aliquots and one formalin-treated control (3.7%, final concentration) were spiked with 14C-leucine (12.2 MBq μmol−1, Sigma, 50 nM final concentration). Samples were incubated in the laboratory at in situ temperature for 1 h in the dark on a shaker. Incorporation was stopped with formalin, and 0.6 ml 50% trichloracetic acid (TCA) was added. Proteins were extracted for 15 min and filtered onto 0.2 μm Nuclepore membranes. Filters were rinsed twice with 1 ml 5% TCA
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and once with 80% ethanol. After dissolving the filters in 0.5 ml Soluene (Packard) and adding 2.5 ml Hionic Fluor (Packard) to each scintillation vial, radioactivity was measured using a Liquid Scintillation Analyzer (2300 TR, Packard). The external standard ratio method was used for quenching. Carbon production was calculated using the equations of Simon and Azam (1989). Production of biofilm bacteria was also estimated with leucine incorporation. Stones of about 1 cm length were transferred to scintillation vials and covered with 4 ml sterile-filtered stream water. Triplicate aliquots and one formalin-treated control (3.7%, final concentration) were spiked with 14C-leucine (5 mM final concentration). After incubation for 1 h under continuous shaking and extraction with TCA on ice, biofilms were removed from stones by ultrasonication for 1 min (20 kHz, 20%; HTU Soni130, Heinemann, Germany). Stones were removed and rinsed, and the supernatant was filtered and measured as described above. To estimate the surface area of the rocks, they were wrapped in tin foil, and the weight of that foil was related to the weight of 1 cm2 foil. Statistical analyses, i.e. the calculation of regression coefficients and p values for relationships between bacterial production and chemical parameters, were performed using the software IBM SPSS Statistics 21.
without phosphorus addition served as a control. In addition, KH2PO4 was added (50 μg P L−1, final concentration) to a second set of sample bottles (triplicates and controls for water and biofilm samples).
2.5. Phosphorus addition experiment
DOC removal ¼
The experiments investigating the effect of phosphorus addition on bacterial production were performed at upstream sites during summer where and when a potential phosphorus limitation was to be expected: upstream site of Rappbode (23 July 2014), upstream site of Hassel (24 July 2014), and Rote Mulde (25 June 2014; see Fig. 2 for TP concentrations in streams). The regular measurement of bacterial production
where BGE is bacterial growth efficiency for calculation of bacterial organic carbon demand (25%; Tranvik, 1988; Weiss and Simon, 1999), and A is the stream surface area in the catchment (dm2). The DOC export from the catchment (kg d−1) was calculated according to Eq. (3):
2.6. Calculation of DOC removal Total areal bacterial production in the streams (μg C dm−2 d−1) was calculated according to Eq. (1): BPTotal ¼ BPWater z þ BPBiofilm
ð1Þ
where BPWater is planktonic BP (μg C L−1 d−1), z is average stream depth (dm), and BPBiofilm is epilithic BP (μg C dm−2 d−1). For Rappbode and Hassel, averages of bacterial production of upstream and downstream sites were calculated. The stream surface area of Hassel in the catchment was estimated from the stream width and length measurements obtained from GIS data (A. Musolff, pers. Comm.). Stream surface area of Hassel was about 0.1% of the entire catchment surface area; a percentage assumed for Rappbode and Rote Mulde as well. DOM removal by stream bacteria in the catchment (kg d− 1) was calculated from total areal bacterial production according to Eq. (2): BPTotal A BGE
DOC export ¼ DOCout Q
ð2Þ
ð3Þ
Fig. 2. Concentrations of total phosphorus (TP; a, b) and dissolved organic carbon (DOC; c, d) concentration at sampling sites in 2013 and 2014. Arrows indicate experiments on phosphorus addition.
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where DOC out is the DOC concentration (mg L−1) at the downstream sites close to the reservoirs, and Q is the discharge (L d−1). It represents the carbon left over from total DOM release after DOM removal in the stream. The total DOC release was calculated as: DOC release ¼ DOC removal þ DOC export:
ð4Þ
It includes allochthonous material from the catchment and autochthonous carbon produced within the stream. DOM removal of the whole stream was then related to the total DOM release, and the ratio of DOM removal to total DOM release was shown as a function of discharge (related to the catchment area, expressed in mm d− 1). 3. Results 3.1. Phosphorus and DOC Concentrations of total phosphorus were high at the upstream sites of Rappbode in April 2013 and of Hassel in November 2013 at high discharge (Fig. 2, Table S1). Usually, the highest values were observed at the downstream site of Hassel, whereas concentrations were low in Rote Mulde. Concentrations of DOC ranged between 2.4–28.3 mg C L−1. In contrast to total phosphorus, they were highest in Rote Mulde, particularly in September 2013 and September 2014. The freshness index of DOM amounted to 0.50–0.69 and showed maximum values almost always at the downstream site of Hassel, while minimum values were observed for Rote Mulde (Fig. 3). The humification index showed a higher variability (5.2–17.8) and was high at the upstream sites of Rappbode and Hassel and in Rote Mulde.
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3.2. Bacterial DOC utilization Planktonic bacterial production ranged between 7 and 387 μg C L− 1 d− 1 and showed seasonal dynamics with low values in spring, maximum activity in summer, and lower production in autumn (Fig. 4). Regarding Rappbode and Hassel, planktonic production was lower at the upstream than at the downstream sites in most cases. The values of Rote Mulde were low in general. Bacterial production of biofilms amounted to 23–1617 μg C dm−2 d−1 and showed similar seasonal dynamics. Considering the production per stream surface area, the proportion of plankton production in the total production depended on water depth and discharge (Table S1): It was almost always lower at shallow upstream sites (where biofilm production dominated) than at deeper downstream sites (where a proportion of plankton production of up to 80% was observed at high discharge). Regarding the control factors of bacterial DOM degradation, both planktonic and biofilm production showed the strongest relationship with temperature (p b 0.001; Fig. 5). In addition, planktonic production was weakly correlated to total phosphorus (p = 0.001), whereas biofilm production was not related to nutrient concentrations at all. Considering measures of DOM quality, planktonic production showed a weak positive relationship to the freshness index (p = 0.004) and a negative one to the humification index (p = 0.001). In contrast, biofilm production was not dependent on DOM quality.
3.3. Phosphorus addition experiment In the nutrient addition experiment, phosphorus stimulated neither planktonic bacterial production nor biofilm bacterial production at the upstream sites of Hassel and Rappbode and in Rote Mulde (Fig. 6).
Fig. 3. Freshness index (β/α; a, b) and humification index (HIX; c, d) of DOM at sampling sites in 2013 and 2014.
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Fig. 4. Planktonic (a, b) and biofilm bacterial production (BP; c, d) at sampling sites (mean ± SD of triplicate water samples for planktonic production and of three stones for biofilm production) in 2013 and 2014.
There was even a tendency towards slightly decreased values after phosphorus addition at the upstream site of Rappbode which was, however, not significant due to unequal variances. 3.4. DOC removal DOM export to reservoirs was highest in spring and autumn of 2013 in Rappbode (Fig. 7) when discharge was high (Table S1). Microbial DOM removal was always lower than DOM export except in July 2013. A similar pattern was observed for Hassel, with higher absolute values for the export compared to Rappbode. In Rote Mulde, the maximum DOM export was measured in autumn 2014. DOM removal was very low (not visible in Fig. 7). The ratio of DOM degradation to total DOC release (removal ratio) was high only at low discharge, namely in summer at high temperatures (Fig. 8). Removal ratios of 75% and 91% were observed in Rappbode and Hassel, respectively, at extremely low discharge in July 2013. In contrast, DOC removal did not exceed DOC export in 2014, and maximum removal ratios of 38% and 45% were found for Hassel and Rappbode, respectively. Nevertheless, the ratio decreased quickly with increasing discharge in all three streams. On an annual average, DOC removal was highest in Hassel and lowest in Rote Mulde (Table 2). Overall, only 0.6–12% of total DOC released by the catchments was degraded within the tributaries. 4. Discussion The measured values in the present study were in the range described in the literature: The concentration of DOC (except for the one high value in the Rote Mulde in September 2014), values of freshness index and humification index of DOM were similar to the respective values in the Bode catchment of which Rappbode and Hassel are sub-
catchments (Kamjunke et al., 2013, 2015). The production of planktonic bacteria were in the same range as those in the river Biobio in Chile (Vargas et al., 2013), in the Bode catchment (Kamjunke et al., 2015) and in streams in Southern Ontario (Williams et al., 2010). The bacterial production in biofilms corresponds to values measured for epilithic bacterial production in small streams in Texas (Scott et al., 2008) and in the Bode catchment (Kamjunke et al., 2015). Temperature was the most important factor controlling both planktonic and biofilm bacterial production what is in accordance with findings of Baldwin et al. (2014). However, only planktonic production was positively related to total phosphorus concentration and freshness index of DOM. In contrast, biofilm production did not show such relationships. These results confirm measurements made in the Bode catchment (Kamjunke et al., 2015), where a dependency of planktonic BP on DOM quality was observed and biofilm BP was independent. The primary production of biofilm algae represents an additional carbon source for biofilm bacteria, and the EPS matrix is regarded as a buffer against changing organic substrate supply and may serve as an energy source during DOC deprivation (Freeman and Lock, 1995). Land use in the catchment did not show a pronounced effect on bacterial production. Bacterial activity was partly dependent on DOM quality which was in turn affected by land use (e.g., high humification index in forest catchments; Kamjunke et al., 2015). However, also low temperature may have limited bacterial production in headwaters. Moreover, DOC was mainly released in the wetlands close to the streams (riparian areas), independent of land use in the catchment (Tittel et al., 2015). The total catchment was important at high discharge only, however, bacterial DOM degradation was high at low discharge (Fig. 8). The proportion of DOM which was degraded by microbial processes was highly dependent on discharge in all three studied streams. We observed that it decreased with increasing discharge. Thus, hypothesis
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Fig. 5. Production of planktonic bacteria and biofilm bacteria as a function of temperature (T), and of planktonic BP as a function of total phosphorus (TP), freshness index (β/α), and humification index (HIX) of DOM.
(2) was confirmed. From our results it can be concluded that DOM degradation was high only under conditions when DOM export to the reservoir was not important (low discharge and high temperature in summer). However, DOM degradation was negligible in comparison to export during periods when the reservoir receives the highest DOM load (high discharge events). We attribute this to short travel times, low temperatures, low bacterioplankton abundances, as well as to the lower significance of biofilm related degradation processes (see above) during high flow situations. Thus, hypothesis (1) has to be
rejected, and we may conclude that the small tributaries of drinking water reservoirs are not important for DOM degradation. This was supported by findings in a temperate river network suggesting that river systems have only a modest capacity to decrease the amounts of terrestrial DOM (Wollheim et al., 2015). Furthermore, bacterial degradation was not enhanced by phosphorus addition. In contrast, enhancement by phosphorus addition was found in some laboratory experiments (Vadstein and Olsen, 1989) and in Amazonian streams (Farjalla et al., 2002). Thus, hypothesis (3) was
Fig. 6. Production of planktonic bacteria and biofilm bacteria with and without addition of phosphorus.
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N. Kamjunke et al. / Science of the Total Environment 548–549 (2016) 51–59 Table 2 Average DOC removal and export in the three catchments. Stream
Rappbode
Hassel
Rote Mulde
DOC removal (kg d−1) DOC export (kg d−1) DOC removal/total DOC release (%)
11.9 85 12.3
14.5 250 5.5
0.7 109 0.6
also not confirmed. In some cases, bacterial production even decreased in the phosphorus treatment. This might be explained by a decreasing percentage of dissolved exudates in total primary production of benthic algae (Baines and Pace, 1991), leading to a declining DOM supply for bacteria. Thus, the tributaries are also not potentially suitable for DOM degradation and improvement of water quality in drinking water reservoirs. 5. Conclusion In contrast to large freshwater systems where the majority of DOM is respired along the way to the ocean, tributaries to drinking water reservoirs in low mountain ranges, which are usually medium-sized streams of short length (b100 km), do not seem to be an important site of microbial DOM degradation. Hydrology and stream length/travel time have to be considered. Thus, the operators of the reservoirs may reduce the DOM load by other measures (e.g., change in land use), or waterworks managers may have to consider advanced techniques in order to remove DOM during the production of drinking water. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2016.01.017. Acknowledgments We thank T. Keller, S. Halbedel, J. Hauschildt und M. Sanchez for help during the field sampling, and A. Hoff and M. Tibke for subsequent analyses in the laboratory. U. Link measured bacterial production, and O. Büttner prepared the map (Fig. 1). The research was funded by the German Federal Ministry of Education and Research (BMBF, project number 02WT1290A). Furthermore, we would like to thank Karsten Rinke and the reviewers for their helpful comments, and Frederic Bartlett for correcting our English. References Fig. 7. Bacterial DOM removal in the streams and export of the remaining DOM into the reservoirs. Please note different scale for y axes.
Fig. 8. Ratio of DOC removal in streams to total DOC release from catchments as a function of discharge. Open symbols represent ratios b0.01 which were not included into correlation.
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Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Bacterial production and their role in the removal of dissolved organic matter from tributaries of drinking water reservoirs Norbert Kamjunke a,b,⁎, Marieke R. Oosterwoud c, Peter Herzsprung b, Jörg Tittel b a b c
Helmholtz-Centre for Environmental Research UFZ, Dept. River Ecology, Brückstraße 3a, D-39114 Magdeburg, Germany Helmholtz-Centre for Environmental Research UFZ, Dept. Lake Research, Brückstraße 3a, D-39114 Magdeburg, Germany Helmholtz-Centre for Environmental Research UFZ, Dept. Hydrogeology, Permoserstr. 15, D-04318 Leipzig, Germany
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• DOM was considerably degraded in summer but not during flood events. • The ratio of DOM removal to total DOM release was negatively related to discharge. • Phosphorus addition did not stimulate bacterial DOM degradation. • A proportion of 0.6–12% of the total DOM was degraded in the tributaries.
a r t i c l e
i n f o
Article history: Received 30 October 2015 Received in revised form 4 January 2016 Accepted 4 January 2016 Available online xxxx Editor: D. Barcelo Keywords: Bacterial production DOC Freshness index Humification index Phosphorus
a b s t r a c t Enhanced concentrations of dissolved organic matter (DOM) in freshwaters are an increasing problem in drinking water reservoirs. In this study we investigated bacterial DOM degradation rates in the tributaries of the reservoirs and tested the hypotheses that (1) DOM degradation is high enough to decrease DOM loads to reservoirs considerably, (2) DOM degradation is affected by stream hydrology, and (3) phosphorus addition may stimulate bacterial DOM degradation. Bacterial biomass production, which was used as a measure of DOM degradation, was highest in summer, and was usually lower at upstream than at downstream sites. An important proportion of bacterial production was realized in epilithic biofilms. Production of planktonic and biofilm bacteria was related to water temperature. Planktonic production weakly correlated to DOM quality and to total phosphorus concentration. Addition of soluble reactive phosphorus did not stimulate bacterial DOM degradation. Overall, DOM was considerably degraded in summer at low discharge levels, whereas degradation was negligible during flood events (when DOM load in reservoirs was high). The ratio of DOM degradation to total DOM release was negatively related to discharge. On annual average, only 0.6–12% of total DOM released by the catchments was degraded within the tributaries. © 2016 Elsevier B.V. All rights reserved.
⁎ Corresponding author at: Helmholtz-Centre for Environmental Research UFZ, Dept. River Ecology, Brückstraße 3a, D-39114 Magdeburg, Germany. E-mail address: [email protected] (N. Kamjunke).
http://dx.doi.org/10.1016/j.scitotenv.2016.01.017 0048-9697/© 2016 Elsevier B.V. All rights reserved.
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1. Introduction Globally, streams and lakes receive an estimated amount of 2.0–2.7 billion tons of terrestrial carbon per year (Cole et al., 2007; Battin et al., 2008; Aufdenkampe et al., 2011). The majority of the terrestrial organic carbon entering freshwater systems is respired to CO2 locally or buried in sediments, whereas only a fraction is discharged into the ocean (Aufdenkampe et al., 2011). The organic carbon in rivers and streams affects aquatic ecosystems and serves as a major energy source for microbes (Findlay et al., 1993; Jonsson et al., 2007). Particularly freshly leached, terrestrial dissolved organic matter (DOM) may influence stream ecosystem processes (Burrows et al., 2013). In addition, the dynamics of DOM mobilization is typically linked to hydrology. About 50% of the DOM was exported by the 10% highest discharges (Hilton et al., 1997). Snowmelt events and heavy rainfall events in summer causing high discharges mobilized the highest DOM fluxes in a small catchment, and spring areas with high DOM pools and pronounced hydrological dynamics were regarded as important sources (Andrews et al., 2011). The concept of hot spots and hot moments (e.g., McClain et al., 2003) may be applied to the DOM export from catchments: Pools of available DOM (e.g., upland areas) are hot spots only during certain periods (hot moments; e.g., heavy rainfall) when hydrological connectivity between the terrestrial sources of DOM and stream discharge is facilitated, enabling a relevant mass flux to surface waters (Casper et al., 2003; Stieglitz et al., 2003). Once the DOM has entered the surface water ecosystem, instream heterotrophic processing of terrestrial derived carbon occurs along the river continuum gradient from the headwater downstream (River Continuum Concept, Vannote et al., 1980). DOM is mainly consumed by heterotrophic bacteria, whereby it is partly removed by respiration and partly transferred to bacterial biomass, the latter process making the carbon available to organisms of higher trophic levels. Terrigenous DOM was shown to be respired by microorganisms rather than incorporated into their biomass (Fashing et al., 2014). Besides bacteria suspended in water, bacteria also grow in biofilms which are substratum-associated consortia of microorganisms including microalgae, bacteria, fungi, protozoans and small metazoans, as well as their extracellular polymeric substances (EPS). Biofilms, e.g. on stones, are regarded as major sites of carbon cycling in streams (Romani et al., 2004; Battin et al., 2008). The use of DOM as a bacterial energy source is controlled by the concentration of limiting nutrients (N and/or P), temperature, and the chemical composition of DOM (Stelzer et al., 2003; Lane et al., 2013). Nutrient enrichment increased biofilm development and reduced the bacterial use of autochthonous carbon within the biofilm (Ziegler and Lyon, 2010). Carbon uptake of biofilms was sensitive to temperature (Baldwin et al., 2014). Land use and vegetation type have a major impact on the composition of DOM, affecting the degree of humification within catchments. DOM in agricultural streams was shown to be more labile and thus more accessible for microbes than DOM in wetland streams (Williams et al., 2010). A positive relationship was found between the aromatic content of DOM and the proportion of forested area in the river network of the Bode catchment in Germany (Kamjunke et al., 2013). Regarding the relationship between DOM quality and bacterial processing, planktonic bacterial production was positively related to labile DOM concentration in streams in Southern Ontario (Williams et al., 2010), and microbial bioavailability of DOM was negatively related to the proportions of humic-like DOM in streams of Maryland (Hosen et al., 2014). Recently, Kamjunke et al. (2015) compared planktonic and biofilm bacterial production with patterns of DOC along a topographic and land-use gradient at 17 sampling sites on one occasion (base flow in late summer). It was found that planktonic production was weakly correlated to the total DOC concentration but strongly to DOM quality. Biofilm production, on the other hand, was independent of both DOC concentration and DOM quality. However, little is known about the effect of stream hydrology on bacterial activity. Bacterial production, measured only during
one investigated period, was found to be low at high discharge in Danube floodplains (Peduzzi et al., 2008). Increasing concentrations of dissolved organic matter in freshwaters due to rising input of organic carbon is a common phenomenon in many regions (Hejzlar et al., 2003; Freeman et al., 2004; Eimers et al., 2008). Monteith et al. (2007) observed such an increase in more than 70% of the investigated surface waters in Northern Europe, Great Britain, and North America. An increase in DOM in English streams of 65% between 1988 and 2000 was found to be particularly caused by DOM from the catchment area, as indicated by the enrichment of humic substances (Freeman et al., 2001); the enhancement was even threefold in the Hudson River (Findlay, 2005). In Germany, a significant increase in DOM concentration was observed in 55% of 86 streams (Sucker et al., 2011). The browning of surface waters leads to increasing problems regarding the production of drinking water. Increasing DOC chlorination cause the formation of mutagenic (Nieuwenhuijsen et al., 2000) and carcinogenic (Sadiq and Rodriguez, 2004) disinfection by-products, taste and odor problems as well as growth of potential pathogens, adversely affect the function of water treatment plants and increase costs for purification of drinking water (Ledesma et al., 2012). The amount of chemicals for flocculation and precipitation rises, the running time of filters decreases, and the amount of sludge in water works increases. The corresponding challenge is to find a cost-effective DOM removal technique which makes use of the instream bacterial production potential at different seasons and along the river-continuum gradient with nutrient addition as a possible management practice. In light of these growing concerns regarding increased DOM inputs into aquatic ecosystems, it is crucial that we understand how much instream bacterial production can contribute to the removal of DOM along a downstream gradient at different seasons and flow conditions. Furthermore, there is a need to explore the feasibility of using nutrient additions (P) as a method of stimulating DOM removal. While one traditional aim of reservoir management is to limit phosphorus imports (Cooke et al., 2005) the mineralization of DOC by bacteria can be limited by phosphorus in particular at high DOC to P ratios (Vadstein and Olsen, 1989). The main goal of the present study is to investigate whether the DOM released from the catchments is transported mainly unchanged by the tributaries to the reservoirs, or if the DOM is degraded considerably within the inflow streams, leaving only a part of the released DOM for the load of the reservoir. If the latter were true, one might consider measures of stimulating DOM degradation to reduce the export to the reservoir. Therefore, we tested the following hypotheses that (1) bacterial DOM degradation rates in the tributaries of the reservoir are high enough to decrease DOM concentrations considerably, (2) DOM degradation is affected by stream hydrology, and (3) phosphate addition may release bacteria from phosphorus limitation and thereby stimulate bacterial DOM degradation. 2. Material and methods 2.1. Study sites and sampling Three streams with different properties were selected: Hassel and Rappbode are tributaries leading to the Rappbode reservoir in the Harz Mountains (Germany; see Rinke et al., 2013; Friese et al., 2014 and Tittel et al., 2015 for detailed description), and Rote Mulde in the Western Ore Mountains is a tributary to the Muldenberg reservoir (see Tittel et al., 2013 for further information; Fig. 1). The Rote Mulde was subjected to a long-term increase in DOC concentration between 1995 and 2010 (Tittel et al., 2013). The drainage area of each catchment/sampling site was determined by delineating the subcatchments on a digital elevation model DEM of 4 m × 4 m resolution using the sampling sites as outlets (Whitebox, QGIS). Topographical maps (Atkis, Landesvermessung und Geobasisinformation Sachsen Anhalt) with a resolution of 1:250,000 were used to determine the contribution of the four land-use classes (agricultural, grassland, forest, urban)
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Fig. 1. Map of the two sampling regions: Muldenberg reservoir with the tributary Rote Mulde, and Rappbode reservoir with the tributaries Rappbode and Hassel. Green: forest, yellow: agricultural area, orange: urban area, red dots: sampling sites.
identified within the catchments. The three catchments differ in land use: 98% forest in the Rote Mulde catchment, some agriculture and 72% forest in the Rappbode catchment, and a dominance of agricultural and urban area with only 37% forest in the Hassel catchment (Fig. 1). As a consequence, different DOC and phosphorus patterns were observed: high DOC/low P in Rote Mulde, low DOC/low P in Rappbode, and low DOC/high P in Hassel (Table 1). Sampling was performed between April and October in 2013 and between March and October in 2014. Hassel and Rappbode were sampled six times a year at an upstream and a downstream site each, whereas Rote Mulde (total length 4 km) was sampled only three times a year and only at the catchments outflow before the reservoir. 2.2. Basic parameters Discharge was measured using a conservative tracer. Depending on discharge, between 200 g and 5000 g of table salt (NaCl) was dissolved in stream water in a bucket and added upstream of the sampling site. Conductivity was measured with multiparameter probes (2013: Hydrolab DSX5, OTT Hydromet; 2014: EXO2, Xylem Inc.), and the area under the conductivity curve was used for discharge calculation. The probes also provided data for water temperature and pH. Water depth was measured for three to seven orthogonal transects within the stream for each site. Total phosphorous (TP) was measured using the ammonium molybdate spectrometric method (see Kamjunke et al., 2013). 2.3. DOC analysis For analysis of dissolved organic carbon (DOC), water samples were transferred into acid-rinsed and combusted brown glass bottles, kept at 4 °C for maximum of 24 h and filtered through glass fiber filters
Table 1 Properties of catchments and streams; chemical values represent ranges during samplings in 2013 and 2014. Stream Catchment area (km2) Mean discharge (L s−1) Total phosphorus (TP, μg L−1) DOC (mg L−1)
Rappbode
Hassel
Rote Mulde
39.1 730 15–40 3.2–5.3
40.5 650 14–86 4.0–9.2
5.4 120 7–28 5.6–28.3
(Whatman GF/F). DOC concentrations were determined after hightemperature combustion (DIMATOC 2000, Dimatec Analysentechnik GmbH, Essen, Germany). Fluorescence excitation emission matrices (EEMs) were collected using a spectrofluorometer (AQUALOG, HORIBA Jobin Yvon, USA). Fluorescence intensity was measured during emission scans (240 nm–600 nm every 3.27 nm; 8 pixel) at set excitation wavelengths in 3 nm increments from 240 nm to 600 nm. A 5 nm bandpass for excitation and emission wavelength and 1 s integration time were used. Fluorescence data were corrected (including blank subtraction) using HORIBA internal software before the correction of inner-filter effects (Kothalawa et al., 2013), a method used in many studies. Afterwards, the EEMF spectra were Rayleigh-scattering masked and corrected for inner-filter effects using an AQUALOG internal recorded UV–Vis absorbance spectrum in the same quartz cell of the fluorescent sample. Two fluorescent indices were used to describe the general DOM structure: freshness index and humification index. The freshness index (ß/α, where ß represents more recently derived DOM and α represents highly decomposed DOM) (Fellman et al., 2010) was computed as the ratio of the emission intensity at 380 nm divided by the highest detected emission intensity between 420 and 435 nm, all obtained for excitation at 310 nm. The ratio of ß/α is an indicator of autochthonous inputs and when applied to our data provides an indicator of relative contribution of recent, microbially produced DOM. The humification index HIX was calculated by dividing the peak area under the emission spectra between 435 and 480 nm with the peak area under the emission spectra between 300 and 345 nm, both at 255 nm excitation wavelength (Zsolnay, 2003). The humification index is associated with the condensing of fluorescence molecules and lower H/C ratios and is therefore an indicator of humicity. 2.4. Bacterial DOC utilization Production of stream water bacteria was measured using the leucine technique (Simon and Azam, 1989) as described by Kamjunke et al. (2015). After storage of samples at 4 °C until the next day, triplicate 5 ml aliquots and one formalin-treated control (3.7%, final concentration) were spiked with 14C-leucine (12.2 MBq μmol−1, Sigma, 50 nM final concentration). Samples were incubated in the laboratory at in situ temperature for 1 h in the dark on a shaker. Incorporation was stopped with formalin, and 0.6 ml 50% trichloracetic acid (TCA) was added. Proteins were extracted for 15 min and filtered onto 0.2 μm Nuclepore membranes. Filters were rinsed twice with 1 ml 5% TCA
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and once with 80% ethanol. After dissolving the filters in 0.5 ml Soluene (Packard) and adding 2.5 ml Hionic Fluor (Packard) to each scintillation vial, radioactivity was measured using a Liquid Scintillation Analyzer (2300 TR, Packard). The external standard ratio method was used for quenching. Carbon production was calculated using the equations of Simon and Azam (1989). Production of biofilm bacteria was also estimated with leucine incorporation. Stones of about 1 cm length were transferred to scintillation vials and covered with 4 ml sterile-filtered stream water. Triplicate aliquots and one formalin-treated control (3.7%, final concentration) were spiked with 14C-leucine (5 mM final concentration). After incubation for 1 h under continuous shaking and extraction with TCA on ice, biofilms were removed from stones by ultrasonication for 1 min (20 kHz, 20%; HTU Soni130, Heinemann, Germany). Stones were removed and rinsed, and the supernatant was filtered and measured as described above. To estimate the surface area of the rocks, they were wrapped in tin foil, and the weight of that foil was related to the weight of 1 cm2 foil. Statistical analyses, i.e. the calculation of regression coefficients and p values for relationships between bacterial production and chemical parameters, were performed using the software IBM SPSS Statistics 21.
without phosphorus addition served as a control. In addition, KH2PO4 was added (50 μg P L−1, final concentration) to a second set of sample bottles (triplicates and controls for water and biofilm samples).
2.5. Phosphorus addition experiment
DOC removal ¼
The experiments investigating the effect of phosphorus addition on bacterial production were performed at upstream sites during summer where and when a potential phosphorus limitation was to be expected: upstream site of Rappbode (23 July 2014), upstream site of Hassel (24 July 2014), and Rote Mulde (25 June 2014; see Fig. 2 for TP concentrations in streams). The regular measurement of bacterial production
where BGE is bacterial growth efficiency for calculation of bacterial organic carbon demand (25%; Tranvik, 1988; Weiss and Simon, 1999), and A is the stream surface area in the catchment (dm2). The DOC export from the catchment (kg d−1) was calculated according to Eq. (3):
2.6. Calculation of DOC removal Total areal bacterial production in the streams (μg C dm−2 d−1) was calculated according to Eq. (1): BPTotal ¼ BPWater z þ BPBiofilm
ð1Þ
where BPWater is planktonic BP (μg C L−1 d−1), z is average stream depth (dm), and BPBiofilm is epilithic BP (μg C dm−2 d−1). For Rappbode and Hassel, averages of bacterial production of upstream and downstream sites were calculated. The stream surface area of Hassel in the catchment was estimated from the stream width and length measurements obtained from GIS data (A. Musolff, pers. Comm.). Stream surface area of Hassel was about 0.1% of the entire catchment surface area; a percentage assumed for Rappbode and Rote Mulde as well. DOM removal by stream bacteria in the catchment (kg d− 1) was calculated from total areal bacterial production according to Eq. (2): BPTotal A BGE
DOC export ¼ DOCout Q
ð2Þ
ð3Þ
Fig. 2. Concentrations of total phosphorus (TP; a, b) and dissolved organic carbon (DOC; c, d) concentration at sampling sites in 2013 and 2014. Arrows indicate experiments on phosphorus addition.
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where DOC out is the DOC concentration (mg L−1) at the downstream sites close to the reservoirs, and Q is the discharge (L d−1). It represents the carbon left over from total DOM release after DOM removal in the stream. The total DOC release was calculated as: DOC release ¼ DOC removal þ DOC export:
ð4Þ
It includes allochthonous material from the catchment and autochthonous carbon produced within the stream. DOM removal of the whole stream was then related to the total DOM release, and the ratio of DOM removal to total DOM release was shown as a function of discharge (related to the catchment area, expressed in mm d− 1). 3. Results 3.1. Phosphorus and DOC Concentrations of total phosphorus were high at the upstream sites of Rappbode in April 2013 and of Hassel in November 2013 at high discharge (Fig. 2, Table S1). Usually, the highest values were observed at the downstream site of Hassel, whereas concentrations were low in Rote Mulde. Concentrations of DOC ranged between 2.4–28.3 mg C L−1. In contrast to total phosphorus, they were highest in Rote Mulde, particularly in September 2013 and September 2014. The freshness index of DOM amounted to 0.50–0.69 and showed maximum values almost always at the downstream site of Hassel, while minimum values were observed for Rote Mulde (Fig. 3). The humification index showed a higher variability (5.2–17.8) and was high at the upstream sites of Rappbode and Hassel and in Rote Mulde.
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3.2. Bacterial DOC utilization Planktonic bacterial production ranged between 7 and 387 μg C L− 1 d− 1 and showed seasonal dynamics with low values in spring, maximum activity in summer, and lower production in autumn (Fig. 4). Regarding Rappbode and Hassel, planktonic production was lower at the upstream than at the downstream sites in most cases. The values of Rote Mulde were low in general. Bacterial production of biofilms amounted to 23–1617 μg C dm−2 d−1 and showed similar seasonal dynamics. Considering the production per stream surface area, the proportion of plankton production in the total production depended on water depth and discharge (Table S1): It was almost always lower at shallow upstream sites (where biofilm production dominated) than at deeper downstream sites (where a proportion of plankton production of up to 80% was observed at high discharge). Regarding the control factors of bacterial DOM degradation, both planktonic and biofilm production showed the strongest relationship with temperature (p b 0.001; Fig. 5). In addition, planktonic production was weakly correlated to total phosphorus (p = 0.001), whereas biofilm production was not related to nutrient concentrations at all. Considering measures of DOM quality, planktonic production showed a weak positive relationship to the freshness index (p = 0.004) and a negative one to the humification index (p = 0.001). In contrast, biofilm production was not dependent on DOM quality.
3.3. Phosphorus addition experiment In the nutrient addition experiment, phosphorus stimulated neither planktonic bacterial production nor biofilm bacterial production at the upstream sites of Hassel and Rappbode and in Rote Mulde (Fig. 6).
Fig. 3. Freshness index (β/α; a, b) and humification index (HIX; c, d) of DOM at sampling sites in 2013 and 2014.
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Fig. 4. Planktonic (a, b) and biofilm bacterial production (BP; c, d) at sampling sites (mean ± SD of triplicate water samples for planktonic production and of three stones for biofilm production) in 2013 and 2014.
There was even a tendency towards slightly decreased values after phosphorus addition at the upstream site of Rappbode which was, however, not significant due to unequal variances. 3.4. DOC removal DOM export to reservoirs was highest in spring and autumn of 2013 in Rappbode (Fig. 7) when discharge was high (Table S1). Microbial DOM removal was always lower than DOM export except in July 2013. A similar pattern was observed for Hassel, with higher absolute values for the export compared to Rappbode. In Rote Mulde, the maximum DOM export was measured in autumn 2014. DOM removal was very low (not visible in Fig. 7). The ratio of DOM degradation to total DOC release (removal ratio) was high only at low discharge, namely in summer at high temperatures (Fig. 8). Removal ratios of 75% and 91% were observed in Rappbode and Hassel, respectively, at extremely low discharge in July 2013. In contrast, DOC removal did not exceed DOC export in 2014, and maximum removal ratios of 38% and 45% were found for Hassel and Rappbode, respectively. Nevertheless, the ratio decreased quickly with increasing discharge in all three streams. On an annual average, DOC removal was highest in Hassel and lowest in Rote Mulde (Table 2). Overall, only 0.6–12% of total DOC released by the catchments was degraded within the tributaries. 4. Discussion The measured values in the present study were in the range described in the literature: The concentration of DOC (except for the one high value in the Rote Mulde in September 2014), values of freshness index and humification index of DOM were similar to the respective values in the Bode catchment of which Rappbode and Hassel are sub-
catchments (Kamjunke et al., 2013, 2015). The production of planktonic bacteria were in the same range as those in the river Biobio in Chile (Vargas et al., 2013), in the Bode catchment (Kamjunke et al., 2015) and in streams in Southern Ontario (Williams et al., 2010). The bacterial production in biofilms corresponds to values measured for epilithic bacterial production in small streams in Texas (Scott et al., 2008) and in the Bode catchment (Kamjunke et al., 2015). Temperature was the most important factor controlling both planktonic and biofilm bacterial production what is in accordance with findings of Baldwin et al. (2014). However, only planktonic production was positively related to total phosphorus concentration and freshness index of DOM. In contrast, biofilm production did not show such relationships. These results confirm measurements made in the Bode catchment (Kamjunke et al., 2015), where a dependency of planktonic BP on DOM quality was observed and biofilm BP was independent. The primary production of biofilm algae represents an additional carbon source for biofilm bacteria, and the EPS matrix is regarded as a buffer against changing organic substrate supply and may serve as an energy source during DOC deprivation (Freeman and Lock, 1995). Land use in the catchment did not show a pronounced effect on bacterial production. Bacterial activity was partly dependent on DOM quality which was in turn affected by land use (e.g., high humification index in forest catchments; Kamjunke et al., 2015). However, also low temperature may have limited bacterial production in headwaters. Moreover, DOC was mainly released in the wetlands close to the streams (riparian areas), independent of land use in the catchment (Tittel et al., 2015). The total catchment was important at high discharge only, however, bacterial DOM degradation was high at low discharge (Fig. 8). The proportion of DOM which was degraded by microbial processes was highly dependent on discharge in all three studied streams. We observed that it decreased with increasing discharge. Thus, hypothesis
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Fig. 5. Production of planktonic bacteria and biofilm bacteria as a function of temperature (T), and of planktonic BP as a function of total phosphorus (TP), freshness index (β/α), and humification index (HIX) of DOM.
(2) was confirmed. From our results it can be concluded that DOM degradation was high only under conditions when DOM export to the reservoir was not important (low discharge and high temperature in summer). However, DOM degradation was negligible in comparison to export during periods when the reservoir receives the highest DOM load (high discharge events). We attribute this to short travel times, low temperatures, low bacterioplankton abundances, as well as to the lower significance of biofilm related degradation processes (see above) during high flow situations. Thus, hypothesis (1) has to be
rejected, and we may conclude that the small tributaries of drinking water reservoirs are not important for DOM degradation. This was supported by findings in a temperate river network suggesting that river systems have only a modest capacity to decrease the amounts of terrestrial DOM (Wollheim et al., 2015). Furthermore, bacterial degradation was not enhanced by phosphorus addition. In contrast, enhancement by phosphorus addition was found in some laboratory experiments (Vadstein and Olsen, 1989) and in Amazonian streams (Farjalla et al., 2002). Thus, hypothesis (3) was
Fig. 6. Production of planktonic bacteria and biofilm bacteria with and without addition of phosphorus.
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N. Kamjunke et al. / Science of the Total Environment 548–549 (2016) 51–59 Table 2 Average DOC removal and export in the three catchments. Stream
Rappbode
Hassel
Rote Mulde
DOC removal (kg d−1) DOC export (kg d−1) DOC removal/total DOC release (%)
11.9 85 12.3
14.5 250 5.5
0.7 109 0.6
also not confirmed. In some cases, bacterial production even decreased in the phosphorus treatment. This might be explained by a decreasing percentage of dissolved exudates in total primary production of benthic algae (Baines and Pace, 1991), leading to a declining DOM supply for bacteria. Thus, the tributaries are also not potentially suitable for DOM degradation and improvement of water quality in drinking water reservoirs. 5. Conclusion In contrast to large freshwater systems where the majority of DOM is respired along the way to the ocean, tributaries to drinking water reservoirs in low mountain ranges, which are usually medium-sized streams of short length (b100 km), do not seem to be an important site of microbial DOM degradation. Hydrology and stream length/travel time have to be considered. Thus, the operators of the reservoirs may reduce the DOM load by other measures (e.g., change in land use), or waterworks managers may have to consider advanced techniques in order to remove DOM during the production of drinking water. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2016.01.017. Acknowledgments We thank T. Keller, S. Halbedel, J. Hauschildt und M. Sanchez for help during the field sampling, and A. Hoff and M. Tibke for subsequent analyses in the laboratory. U. Link measured bacterial production, and O. Büttner prepared the map (Fig. 1). The research was funded by the German Federal Ministry of Education and Research (BMBF, project number 02WT1290A). Furthermore, we would like to thank Karsten Rinke and the reviewers for their helpful comments, and Frederic Bartlett for correcting our English. References Fig. 7. Bacterial DOM removal in the streams and export of the remaining DOM into the reservoirs. Please note different scale for y axes.
Fig. 8. Ratio of DOC removal in streams to total DOC release from catchments as a function of discharge. Open symbols represent ratios b0.01 which were not included into correlation.
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