Development of a Rapid Assimilable Organic Carbon Method for Water

A rapid method for measurement of assimilable organic carbon (AOC) is proposed. The time needed to perform the assay is reduced by increasing the incubation temperature and increasing the inoculum density. The ATP luciferin-luciferase method quickly enumerates the test organisms without the need for plate count media or dilution bottles. There was no significant difference between AOC values determined with strain P17 for the ATP and plate count procedures. For strain NOX, the plate count procedure underestimated bacterial levels in some samples. Comparison of AOC values obtained by the Belleville laboratory (by the ATP technique) and the Stroud Water Research Center (by plate counts) showed that values were significantly correlated and not significantly different. The study concludes that the rapid AOC method can quickly determine the bacterial growth potential of water within 2 to 4 days.     

Characterization of Bioluminescent Derivatives of Assimilable Organic Carbon Test Bacteria

The assimilable organic carbon (AOC) test is a standardized measure of the bacterial growth potential of treated water. We describe the design and initial development of an AOC assay that uses bioluminescent derivatives of AOC test bacteria. Our assay is based on the observation that bioluminescence peaks at full cell yield just prior to the onset of the stationary phase during growth in a water sample. Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX bacteria were mutagenized with luxCDABE operon fusion and inducible transposons and were selected on minimal medium. Independent mutants were screened for high luminescence activity and predicted AOC assay sensitivity. All mutants tested were able to grow in tap water under AOC assay conditions. Strains P-17 I5 (with p-aminosalicylate inducer) and NOX I3 were chosen for use in the bioluminescence AOC test. Peak bioluminescence and plate count AOC were linearly related for both test bacteria, though data suggest that the P-17 bioluminescence assay requires more consistent luminescence monitoring. Bioluminescence results were obtained 2 or 3 days postinoculation, compared with 5 days for the ATP luminescence AOC assay and 8 days for the plate count assay. Plate count AOC assay results for nonmutant and bioluminescent bacteria from 36 water samples showed insignificant differences, indicating that the luminescent bacteria retained a full range of AOC measurement capability. This bioluminescence method is amenable to automation with a microplate format with programmable reagent injection.     

Effects of Assimilable Organic Carbon and Free Chlorine on Bacterial Growth in Drinking Water

Assimilable organic carbon (AOC) is one of the most important factors affecting the re-growth of microorganisms in drinking water. High AOC concentrations result in biological instability, but disinfection kills microbes to ensure the safety of drinking water. Free chlorine is an important oxidizing agent used during the disinfection process. Therefore, we explored the combined effects of AOC and free chlorine on bacterial growth in drinking water using flow cytometry (FCM). The initial AOC concentration was 168 μg.L^-1 in all water samples. Without free chlorine, the concentrations of intact bacteria increased but the level of AOC decreased. The addition of sodium hypochlorite caused an increase and fluctuation in AOC due to the oxidation of organic carbon. The concentrations of intact bacteria decreased from 1.1×10⁵ cells.mL^-1 to 2.6×10⁴ cells.mL^-1 at an initial free chlorine dose of 0.6 mg.L^-1 to 4.8×10⁴ cells.mL^-1 at an initial free chlorine dose of 0.3 mg.L^-1 due to free chlorine originating from sodium hypochlorite. Additionally, free chlorine might be more obviously affected AOC concentrations than microbial growth did. These results suggested that AOC and free chlorine might have combined effects on microbial growth. In this study, our results showed concentrations determined by FCM were higher than those by HPC, which indicated that some E. coli detected by FCM might not be detected using HPC in drinking water. The level of free chlorine might restrain the consumption of AOC by inhibiting the growth of E. coli; on the other hand, chlorination might increase the level of AOC, thereby increase the potential for microbial growth in the drinking water network.     

Kinetics and Yields of Pesticide Biodegradation at Low Substrate Concentrations and under Conditions Restricting Assimilable Organic Carbon

The fundamentals of growth-linked biodegradation occurring at low substrate concentrations are poorly understood. Substrate utilization kinetics and microbial growth yields are two critically important process parameters that can be influenced by low substrate concentrations. Standard biodegradation tests aimed at measuring these parameters generally ignore the ubiquitous occurrence of assimilable organic carbon (AOC) in experimental systems which can be present at concentrations exceeding the concentration of the target substrate. The occurrence of AOC effectively makes biodegradation assays conducted at low substrate concentrations mixed-substrate assays, which can have profound effects on observed substrate utilization kinetics and microbial growth yields. In this work, we introduce a novel methodology for investigating biodegradation at low concentrations by restricting AOC in our experiments. We modified an existing method designed to measure trace concentrations of AOC in water samples and applied it to systems in which pure bacterial strains were growing on pesticide substrates between 0.01 and 50 mg liter⁻¹. We simultaneously measured substrate concentrations by means of high-performance liquid chromatography with UV detection (HPLC-UV) or mass spectrometry (MS) and cell densities by means of flow cytometry. Our data demonstrate that substrate utilization kinetic parameters estimated from high-concentration experiments can be used to predict substrate utilization at low concentrations under AOC-restricted conditions. Further, restricting AOC in our experiments enabled accurate and direct measurement of microbial growth yields at environmentally relevant concentrations for the first time. These are critical measurements for evaluating the degradation potential of natural or engineered remediation systems. Our work provides novel insights into the kinetics of biodegradation processes and growth yields at low substrate concentrations.     

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 μm 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.     

Uptake of Dissolved Organic Carbon by Gammaproteobacterial Subgroups in Coastal Waters of the West Antarctic Peninsula

Heterotrophic bacteria are well known to be key players in the turnover of dissolved organic material (DOM) in the oceans, but the relationship between DOM uptake and bacterial clades is still not well understood. Here we explore the turnover and single-cell use of glucose, an amino acid mixture, N-acetylglucosamine (NAG), and protein by gammaproteobacterial clades in coastal waters of the West Antarctic Peninsula in summer and fall. More than 60% of the cells within two closely related gammaproteobacterial clades, Ant4D3 and Arctic96B-16, were active in using the amino acid mixture, protein, and NAG. In contrast, an average of only 7% of all SAR86 cells used amino acids and protein even in summer when DOM use was high. In addition to DOM uptake within a group, we explored the contribution of the three gammaproteobacterial groups to total community uptake of a compound. SAR86 contributed 5- to 10-fold less than the other gammaproteobacterial subgroups to the uptake of all compounds. We found that the overall contribution of the Ant4D3 clade to DOM uptake was highest, whereas the SAR86 clade contributed the least to DOM turnover in West Antarctic Peninsula waters. Our results suggest that the low growth activity of a bacterial clade leads to low abundance, fewer active cells and a low contribution to the turnover of DOM components.     

Organic Carbon in a Eutrophic Fishpond

Accumulation of dissolved organic carbon (DOC) was studied in a eutrophic pond and in cultures of Scenedesmus abundans. Samples of pond water and media were treated with a cation exchanger in Na cycle and chromatographed on Sephadex G 25 and G 10. — UV spectra of the peak fractions were recorded. In the pond about half of the organic carbon is in fractions with an apparent molecular weight (AMW) of 500–1000, while in the algal medium (after cultivation) most of organic carbon has an AMW of about 120. These and spectral data did not suggest that aromatics and compounds with conjugated double bonds were major organic constituents of DOC.     

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-μm 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.     

Response of Bacterial Metabolic Activity to Riverine Dissolved Organic Carbon and Exogenous Viruses in Estuarine and Coastal Waters: Implications for CO2 Emission

A cross-transplant experiment between estuarine water and seawater was conducted to examine the response of bacterial metabolic activity to riverine dissolved organic carbon (DOC) input under virus-rich and virus-free conditions, as well as to exogenous viruses. Riverine DOC input increased bacterial production significantly, but not bacterial respiration (BR) because of its high lability. The bioavailable riverine DOC influenced bulk bacterial respiration in two contrasting ways; it enhanced the bulk BR by stimulating bacterial growth, but simultaneously reduced the cell-specific BR due to its high lability. As a result, there was little stimulation of the bulk BR by riverine DOC. This might be partly responsible for lower CO₂ degassing fluxes in estuaries receiving high sewage-DOC that is highly labile. Viruses restricted microbial decomposition of riverine DOC dramatically by repressing the growth of metabolically active bacteria. Bacterial carbon demand in the presence of viruses only accounted for 7–12% of that in the absence of viruses. Consequently, a large fraction of riverine DOC was likely transported offshore to the shelf. In addition, marine bacteria and estuarine bacteria responded distinctly to exogenous viruses. Marine viruses were able to infect estuarine bacteria, but not as efficiently as estuarine viruses, while estuarine viruses infected marine bacteria as efficiently as marine viruses. We speculate that the rapid changes in the viral community due to freshwater input destroyed the existing bacteria-virus relationship, which would change the bacterial community composition and affect the bacterial metabolic activity and carbon cycling in this estuary.     

Carbon Mineralization and Labile Organic Carbon Pools in the Sandy Soils of a North Florida Watershed

The large pool of actively cycling carbon (C) held in soils is susceptible to release due to changes in landuse, management, or climate. Yet, the amount and distribution of potentially mineralizable C present in soils of various types and the method by which this soil C fraction can best be quantified, are not well established. The distribution of total organic C (TOC), extractable C pools (hot-water-extractable and acid-hydrolyzable), and in vitro mineralizable C in 138 surface soils across a north Florida watershed was found to be quite heterogeneous. Thus, these C quality parameters could not statistically distinguish the eight landuses or four major soil orders represented. Only wetland and upland forest soils, with the largest and smallest C pool size, respectively, were consistently different from the soils of other landuse types. Variations in potential C mineralization were best explained by TOC (62%) and hot-water-extractable C (59%), whereas acid-hydrolyzable C (32%) and clay content (35%) were generally not adequate indicators of C bioavailability. Within certain landuse and soil orders (Alfisol, Wetland and Rangeland, all with >3% clay content), however, C mineralization and clay content were directly linearly correlated, indicating a possible stimulatory effect of clay on microbial processing of C. Generally, the sandy nature of these surface soils imparted a lack of protection against C mineralization and likely resulted in the lack of landuse/soil order differences in the soil C pools. If a single parameter is to be chosen to quantify the potential for soil C mineralization in southeastern U.S. coastal plain soils, we recommend TOC as the most efficient soil variable to measure. Author Contributions  Conceived of or designed study: Sabine Grunwald, Nick Comerford, and James Sickman—Performed research: Mi-Youn Ahn—Analyzed data: Mi-Youn Ahn, Andrew Zimmerman, and Nick Comerford—Contributed new methods or models: Andrew Zimmerman, Nick Comerford, and James Sickman—Wrote the paper: Mi-Youn Ahn, Andrew Zimmerman, and Nick Comerford.     

Microbiosensors for Measurement of Microbially Available Dissolved Organic Carbon: Sensor Characteristics and Preliminary Environmental Application

Microbial respiration-based microbiosensors used for quantification of available dissolved organic carbon (ADOC) instantaneously respired by microorganisms are described. The sensing membranes contained aerobic seawater microorganisms immobilized in a polyurethane hydrogel. Molecular investigations revealed that the bacterial strain used was most closely related to Staphylococcus warneri. This strain was characterized by low substrate selectivity, which was reflected in the response to various mono- and disaccharides, short-chain fatty acids, and amino acids, as determined using Biolog microplates. Specific emphasis was placed on critically assessing biosensor functioning that was affected by preconditioning of the selected bacterial strain, chemical and geometric properties of the sensing membrane (e.g., composition, permeability, and thickness), and the distribution, biomass, and physiological state of immobilized cells, as well as the exposure conditions (e.g., temperature and nutrient supply). The sensors revealed that there was a linear response up to a glucose concentration of 500 μM depending on the type, characteristics, and recent history of the sensors. The detection limit of the sensors was equivalent to about 6 to 10 μM glucose. The 90% response time ranged from 1 to 5 min. Generally, the response of the biosensors became weaker with time. The shelf lives of individual sensors were up to 2 weeks. Measurements based on optical ADOC microbiosensors revealed that in photoautotrophically dominated sandy coastal sediments, the pool sizes and turnover of ADOC were regulated by the photosynthetic activity of benthic microalgae and microbial aerobic respiration. A large increase in ADOC production was observed shortly after the microphytobenthic primary production reached the maximum value at midday, whereas ADOC was consumed by microbial respiration during the night.     

Dissolved Organic Carbon in Terrestrial Ecosystems: Synthesis and a Model

The movement of dissolved organic carbon (DOC) through soils is an important process for the transport of carbon within ecosystems and the formation of soil organic matter. In some cases, DOC fluxes may also contribute to the carbon balance of terrestrial ecosystems; in most ecosystems, they are an important source of energy, carbon, and nutrient transfers from terrestrial to aquatic ecosystems. Despite their importance for terrestrial and aquatic biogeochemistry, these fluxes are rarely represented in conceptual or numerical models of terrestrial biogeochemistry. In part, this is due to the lack of a comprehensive understanding of the suite of processes that control DOC dynamics in soils. In this article, we synthesize information on the geochemical and biological factors that control DOC fluxes through soils. We focus on conceptual issues and quantitative evaluations of key process rates to present a general numerical model of DOC dynamics. We then test the sensitivity of the model to variation in the controlling parameters to highlight both the significance of DOC fluxes to terrestrial carbon processes and the key uncertainties that require additional experiments and data. Simulation model results indicate the importance of representing both root carbon inputs and soluble carbon fluxes to predict the quantity and distribution of soil carbon in soil layers. For a test case in a temperate forest, DOC contributed 25% of the total soil profile carbon, whereas roots provided the remainder. The analysis also shows that physical factors—most notably, sorption dynamics and hydrology—play the dominant role in regulating DOC losses from terrestrial ecosystems but that interactions between hydrology and microbial–DOC relationships are important in regulating the fluxes of DOC in the litter and surface soil horizons. The model also indicates that DOC fluxes to deeper soil layers can support a large fraction (up to 30%) of microbial activity below 40 cm. Received 14 January 2000; accepted 6 September 2000     

酚氧化酶试纸条制备与应用

制备出一种酚氧化酶试纸条,于３７℃６ｈ内可完成酵母样真菌的酚氧化酶试验,较常规培养基试验法至少能提前 １８～２４ｈ获得结果。该纸条置４℃保存,有效期６个月,对鉴定酵母样真菌具有快速、准确、实用性。     

Temperature Dependence of Photodegradation of Dissolved Organic Matter to Dissolved Inorganic Carbon and Particulate Organic Carbon

Photochemical transformation of dissolved organic matter (DOM) has been studied for more than two decades. Usually, laboratory or “in-situ” experiments are used to determine photodegradation variables. A common problem with these experiments is that the photodegradation experiments are done at higher than ambient temperature. Five laboratory experiments were done to determine the effect of temperature on photochemical degradation of DOM. Experimental results showed strong dependence of photodegradation on temperature. Mathematical modeling of processes revealed that two different pathways engaged in photochemical transformation of DOM to dissolved inorganic carbon (DIC) strongly depend on temperature. Direct oxidation of DOM to DIC dominated at low temperatures while conversion of DOM to intermediate particulate organic carbon (POC) prior to oxidation to DIC dominated at high temperatures. It is necessary to consider this strong dependence when the results of laboratory experiments are interpreted in regard to natural processes. Photodegradation experiments done at higher than ambient temperature will necessitate correction of rate constants.     

Leaf Litter as a Source of Dissolved Organic Carbon in Streams

Dissolved organic carbon (DOC) is an abundant form of organic matter in stream ecosystems. Most research has focused on the watershed as the source of DOC in streams, but DOC also comes from leaching of organic matter stored in the stream channel. We used a whole-ecosystem experimental approach to assess the significance of leaching of organic matter in the channel as a source of DOC in a headwater stream. Inputs of leaf litter were excluded from a forested Appalachian headwater stream for 3 years. Stream-water concentration, export, and instream generation of DOC were reduced in the litter-excluded stream as compared with a nearby untreated reference stream. The proportion of high molecular weight (HMW) DOC (more than 10,000 daltons) in stream water was not altered by litter exclusion. Mean DOC concentration in stream water was directly related to benthic leaf-litter standing stock. Instream generation of DOC from leaf litter stored in the stream channel contributes approximately 30% of daily DOC exports in this forested headwater stream. This source of DOC is greatest during autumn and winter and least during spring and summer. It is higher during increasing discharge than during base flow. We conclude that elimination of litter inputs from a forested headwater stream has altered the biogeochemistry of DOC in this ecosystem. Received 2 September 1997; accepted 27 January 1998.     

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.     

Carbon Dioxide in Boreal Surface Waters: A Comparison of Lakes and Streams

The quantity of carbon dioxide (CO₂) emissions from inland waters into the atmosphere varies, depending on spatial and temporal variations in the partial pressure of CO₂ (pCO₂) in waters. Using 22,664 water samples from 851 boreal lakes and 64 boreal streams, taken from different water depths and during different months we found large spatial and temporal variations in pCO₂, ranging from below atmospheric equilibrium to values greater than 20,000???atm with a median value of 1048???atm for lakes (n?=?11,538 samples) and 1176???atm for streams (n?=?11,126). During the spring water mixing period in April/May, distributions of pCO₂ were not significantly different between stream and lake ecosystems (P?>?0.05), suggesting that pCO₂ in spring is determined by processes that are common to lakes and streams. During other seasons of the year, however, pCO₂ differed significantly between lake and stream ecosystems (P?<?0.0001). The variable that best explained the differences in seasonal pCO₂ variations between lakes and streams was the temperature difference between bottom and surface waters. Even small temperature differences resulted in a decline of pCO₂ in lake surface waters. Minimum pCO₂ values in lake surface waters were reached in July. Towards autumn pCO₂ strongly increased again in lake surface waters reaching values close to the ones found in stream surface waters. Although pCO₂ strongly increased in the upper water column towards autumn, pCO₂ in lake bottom waters still exceeded the pCO₂ in surface waters of lakes and streams. We conclude that throughout the year CO₂ is concentrated in bottom waters of boreal lakes, although these lakes are typically shallow with short water retention times. Highly varying amounts of this CO₂ reaches surface waters and evades to the atmosphere. Our findings have important implications for up-scaling CO₂ fluxes from single lake and stream measurements to regional and global annual fluxes.     

Second-Order Model to Predict Microbial Degradation of Organic Compounds in Natural Waters

The reliability of second-order rate constants for assessing microbial degradation kinetics in natural waters was examined by using three compounds that undergo hydrolytic degradation. The butoxyethyl ester of 2,4-dichlorophenoxyacetic acid was studied in water samples from 31 sites, malathion was examined in water from 14 sites, and chlorpropham was studied in samples from 11 sites. The coefficient of variation for rate constants for each compound was less than 65% over all sites. Additional studies indicated that the rate conformed to second-order kinetics; that is, the rate was proportional to both bacterial and xenobiotic concentrations.