Complexity of dissolved organic matter in the molecular size dimension: insights from coupled size exclusion chromatography electrospray ionisation mass spectrometry.

This paper investigates the relationship between apparent size distribution and molecular complexity of dissolved organic matter from the natural environment. We used a high pressure size exclusion chromatography (HPSEC) method coupled to UV-Vis diode array detection (UV-DAD) and electrospray ionisation mass spectrometry (ESI-MS) in order to compare the apparent size of natural organic matter, determined by HPSEC-UV and the molecular mass determined online by ESI-MS. We found that there was a clear discrepancy between the two methods, and found evidence for an important pool of organic matter that has a strong UV absorbance and no ESI-MS signal. Contrary to some previous research, we found no evidence that apparently high molecular weight organic matter is constituted by aggregates of low molecular weight (<1000 Da) material. Furthermore, our results suggest that the majority of apparent size variability within the ESI ionisable pool of organic matter is due to secondary interaction and exclusion effects on the HPSEC column, and not true differences in hydrodynamic size or intermolecular aggregation.

Tracking changes in the optical properties and molecular composition of dissolved organic matter during drinking water production.

Absorbance, 3D fluorescence and ultrahigh resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) were used to explain patterns in the removal of chromophoric and fluorescent dissolved organic matter (CDOM and FDOM) at the molecular level during drinking water production at four large drinking water treatment plants in Sweden. When dissolved organic carbon (DOC) removal was low, shifts in the dissolved organic matter (DOM) composition could not be detected with commonly used DOC-normalized parameters (e.g. specific UV254 absorbance - SUVA), but was clearly observed by using differential absorbance and fluorescence or ESI-FT-ICR-MS. In addition, we took a novel approach by identifying how optical parameters were correlated to the elemental composition of DOM by using rank correlation to connect optical properties to chemical formulas assigned to mass peaks from FT-ICR-MS analyses. Coagulation treatment selectively removed FDOM at longer emission wavelengths (450-600 nm), which significantly correlated with chemical formulas containing oxidized carbon (average carbon oxidation state ≥ 0), low hydrogen to carbon ratios (H/C: average ± SD = 0.83 ± 0.13), and abundant oxygen-containing functional groups (O/C = 0.62 ± 0.10). Slow sand filtration was less efficient in removing DOM, yet selectively targeted FDOM at shorter emission wavelengths (between 300 and 450 nm), which commonly represents algal rather than terrestrial sources. This shorter wavelength FDOM correlated with chemical formulas containing reduced carbon (average carbon oxidation state ≤ 0), with relatively few carbon-carbon double bonds (H/C = 1.32 ± 0.16) and less oxygen per carbon (O/C = 0.43 ± 0.10) than those removed during coagulation. By coupling optical approaches with FT-ICR-MS to characterize DOM, we were for the first time able to confirm the molecular composition of absorbing and fluorescing DOM selectively targeted during drinking water treatment.

Simultaneous measurements of organic carbon mineralization and bacterial production in oxic and anoxic lake sediments.

Based on work in marine sediments it can be hypothesized that (i) overall OM mineralization depends on the enzymatic capacity and is largely independent from the energy yield, (ii) similar oxic and anoxic rates are expected for fresh OM, while oxic rates should be faster for old OM that is partially degraded or adsorbed to particles, and (iii) that the thermodynamic energy yield does not regulate mineralization, but primarily determines the energy fraction allocated to bacterial production (BP). We addressed these hypotheses by simultaneous measurements of mineralization rates (MR) and BP in sediments from a eutrophic lake, along with MR measurements in sediments of a dystrophic lake. Anoxic MR were 44 and 78% of oxic MR in the eutrophic and dystrophic lake, respectively, which was always higher than expected given the theoretical energy yields. The BP:MR ratio was 0.94 and 0.24 in the oxic and anoxic treatments, respectively, in accordance with the expected energy yields. Thus, the results support all three hypotheses above. We also critically discuss BP measurements in sediments and suggest that bacterial growth efficiency values from simultaneous MR and BP measurements can be used to evaluate the reliability of BP estimates.

Antagonism between bacteria and fungi on decomposing aquatic plant litter.

Bacterial and fungal decomposers of aquatic plant litter may exhibit either synergistic or antagonistic interactions, which are likely to influence microbial growth as well as the decomposition of litter and, eventually, the carbon metabolism of aquatic systems. To elucidate such interactions, we inoculated decomposing Phragmites culms in microcosms with fungal isolates and with natural communities of bacteria and fungi in different combinations. The development of fungal and bacterial biomass and the carbon dynamics were studied during several months of degradation. The results show a bilateral antagonistic relationship between bacteria and fungi. After 3 months, fungal biomass accumulation was approximately 12 times higher in the absence than in the presence of bacteria. Bacterial biomass accumulation was about double in the absence of fungi compared to when fungi were present. Similar interactions developed between a natural assemblage of bacteria and five different fungal strains isolated from Phragmites litter (three identified hyphomycetes and two unidentified strains). Despite the great difference in biomass development between the treatments, the carbon metabolism was similar regardless of whether fungi and/or bacteria were present alone or in coexistence. We suggest that the antagonism between bacteria and fungi is an important controlling factor for microbial colonization and growth on aquatic plant litter.

Large differences in the fraction of active bacteria in plankton, sediments, and biofilm.

Generally, only a small fraction of free-living pelagic bacteria are metabolically active, while particle-associated bacteria usually exhibit a larger proportion of active bacteria. Most previous studies on the active fraction of bacteria focus on planktonic communities, and there are only a few studies on sediment and epiphytic biofilm bacteria. We compared the active fraction of the total number of bacteria in three different habitats of the littoral zone of Lake Erken, Sweden, including the sediments, the epiphytic biofilm on the submerged macrophyte Ranunculus circinatus, and the water column. Active bacteria were detected as those with an active electron transport system, identified by the capacity to reduce the tetrazolium salt CTC (5-cyano-2,3-ditolyltetrazolium chloride) into its fluorescent, water insoluble state. There were large differences between habitats. The active fraction of the total number of bacteria detected by fluorescence microscopy (annual mean +/- SD) in the sediments was 46 +/- 10%, on R. circinatus 37 +/- 18%, and in the water column 4 +/- 4%. The abundance of CTC-reducing cells was correlated with total bacterial abundance, and the fraction of CTC-reducing bacteria generally increased with total bacterial abundance, for all the habitats. Consequently, the difference in the fraction of CTC-reducing bacteria between the habitats could be attributed to different densities of bacteria, with a larger proportion of active bacteria at higher bacterial densities.

The leucine incorporation method estimates bacterial growth equally well in both oxic and anoxic lake waters.

Bacterial biomass production is often estimated from incorporation of radioactively labeled leucine into protein, in both oxic and anoxic waters and sediments. However, the validity of the method in anoxic environments has so far not been tested. We compared the leucine incorporation of bacterial assemblages growing in oxic and anoxic waters from three lakes differing in nutrient and humic contents. The method was modified to avoid O(2) contamination by performing the incubation in syringes. Isotope saturation levels in oxic and anoxic waters were determined, and leucine incorporation rates were compared to microscopically observed bacterial growth. Finally, we evaluated the effects of O(2) contamination during incubation with leucine, as well as the potential effects of a headspace in the incubation vessel. Isotope saturation occurred at a leucine concentration of above about 50 nM in both oxic and anoxic waters from all three lakes. Leucine incorporation rates were linearly correlated to observed growth, and there was no significant difference between oxic and anoxic conditions. O(2) contamination of anoxic water during 1-h incubations with leucine had no detectable impact on the incorporation rate, while a headspace in the incubation vessel caused leucine incorporation to increase in both anoxic and O(2)-contaminated samples. The results indicate that the leucine incorporation method relates equally to bacterial growth rates under oxic and anoxic conditions and that incubation should be performed without a headspace.

Effects of enrichment on simple aquatic food webs.

Simple models, based on Lotka-Volterra types of interactions between predator and prey, predict that enrichment will have a destabilizing effect on populations and that equilibrium population densities will change at the top trophic level and every second level below. We experimentally tested these predictions in three aquatic food web configurations subjected to either high or low nutrient additions. The results were structured by viewing the systems as either food chains or webs and showed that trophic level biomass increased with enrichment, which contradicts food chain theory. However, within each trophic level, food web configuration affected the extent to which different functional groups responded to enrichment. By dividing trophic levels into functional groups, based on vulnerability to consumption, we were able to identify significant effects that were obscured when systems were viewed as food chains. The results support the prediction that invulnerable prey may stabilize trophic-level dynamics by replacing other, more vulnerable prey. Furthermore, the vulnerable prey, such as Daphnia and edible algae, responded as predicted by the paradox of enrichment hypothesis; that is, variability in population density increased with enrichment. Hence, by describing ecosystems as a matrix of food web interactions, and by recognizing the interplay between interspecific competition and predation, a more complete description of the ecosystem function was obtained compared to when species were placed into distinct trophic levels.

Colloidal and Dissolved Organic Matter Excreted by a Mixotrophic Flagellate during Bacterivory and Autotrophy.

Excretion of dissolved and colloidal organic carbon by a mixotrophic flagellate, the chrysophyte Poterioochromonas malhamensis, was studied. Flagellates were incubated either with C-labeled bacteria or with inorganic C, in order to compare organic exudates originating from primary production with exudates originating from ingested bacteria. Colloids of >0.02 mum constituted a larger fraction of the exudates originating from ingested bacteria, compared with exudates derived from primary production. Flagellate feeding on bacteria specifically labeled in different cell components was compared. Cell wall components gave rise to less colloidal organic carbon than did other cell constituents. To investigate the degradability of flagellate C-exudates, they were added to lake water and mineralization to CO(2) was monitored. Bacterially derived exudates were more recalcitrant than exudates originating from photosynthesis. The results support the hypothesis that bacterial utilization of labile organic compounds, followed by flagellate bacterivory and exudation, results in a transformation of labile organic matter into more recalcitrant forms.

Bacterioplankton growth on fractions of dissolved organic carbon of different molecular weights from humic and clear waters.

The ability of fractions of dissolved organic carbon (DOC) of different molecular weights (MW) to support bacterial growth was studied in batch culture experiments. Natural pelagic bacteria were inoculated into particle-free (0.2-mum filtered) water, taken from 10 oligotrophic lakes of differing humic content, and either used without further treatments or ultrafiltered to remove DOC of >10,000 MW or >1,000 MW. Stationaryphase abundance of bacteria in the cultures was used as an estimate of bacterial carrying capacity. High-MW DOC (>10,000) comprised an increasing fraction of total DOC with increasing total DOC and humic content of the lakes. High-MW DOC was generally more available to bacteria (i.e., more bacteria were produced per unit of organic carbon initially present) than low-MW (<10,000) DOC. The availability to bacteria of this high-MW DOC decreased with increasing humic content. However, although less available in humic lakes than in clearwater lakes, the higher abundance of high-MW DOC made it quantitatively more important as a bacterial substrate; i.e., a larger fraction of the total bacterial yield of the cultures was due to high-MW DOC compounds in humic lakes than in clearwater lakes. On the average, 48% of bacterial growth occurred at the expense of DOC of <10,000 MW. DOC of <1,000 MW was responsible for an average of 22% of bacterial growth, with no significant correlation to humic content and DOC concentration of lakes. The DOC which supports bacterial growth, as well as the total DOC, is of different quality in humic and clearwater lakes.

Microbial degradation of xenobiotic, aromatic pollutants in humic water.

The microbial degradation of a number of 14C-labeled, recalcitrant, aromatic pollutants, including trichloroguaiacol and di-, tri-, and pentachlorophenol, was investigated in aquatic model systems in the laboratory. Natural, mixed cultures of microorganisms in the water from a brown-water lake with a high content of humic compounds mineralized all of the tested substances to a higher degree than did microorganisms in the water from a clear-water lake. Dichlorophenol was the most rapidly degraded pollutant.

Availability of dissolved organic carbon for planktonic bacteria in oligotrophic lakes of differing humic content.

Bacterioplankton from 10 oligotrophic lakes, representing a gradient from clearwater to polyhumic, were grown in dilution cultures of sterile filtered lake water. The bacterial biomass achieved in the stationary phase of the dilution cultures was positively correlated with the amount of both humic matter and dissolved organic carbon (DOC) in the lakes. About the same fraction of the total DOC pool was consumed in the dilution cultures of all lakes (average 9.5%, coefficient of variation (CV) 24%), with approximately the same growth efficiency (average 26%, CV 28%). Thus, humic lakes could support a higher bacterial biomass than clearwater lakes due to their larger DOC pools. The relevance of the results to planktonic food webs of humic and clearwater lakes is discussed.

Bacterial growth in mixed cultures on dissolved organic carbon from humic and clear waters.

Interactions between bacterial assemblages and dissolved organic carbon (DOC) from different sources were investigated. Mixed batch cultures were set up with water from a humic and a clear-water lake by a 1:20 dilution of the bacterial assemblage (1.0 mum of prefiltered lake water) with natural medium (sterile filtered lake water) in all four possible combinations of the two waters and their bacterial assemblages. Bacterial numbers and biomass, DOC, thymidine incorporation, ATP, and uptake of glucose and phenol were followed in these cultures. Growth curves and exponential growth rates were similar in all cultures, regardless of inoculum or medium. However, bacterial biomass produced was double in cultures based on water from the humic lake. The fraction of DOC consumed by heterotrophic bacteria during growth was in the same range, 15 to 22% of the total DOC pool, in all cultures. Bacterial growth efficiency, calculated from bacterial biomass produced and DOC consumed, was in the order of 20%. Glucose uptake reached a peak during exponential growth in all cultures. Phenol uptake was insignificant in the cultures based on the clear-water medium, but occurred in humic medium cultures after exponential growth. The similarity in the carbon budgets of all cultures indicated that the source of the bacterial assemblage did not have a significant effect on the overall carbon flux. However, fluxes of specific organic compounds differed, as reflected by glucose and phenol uptake, depending on the nature of the DOC and the bacterial assemblage.