Comparison of Acridine Orange, Acriflavine, and Bisbenzimide Stains for Enumeration of Bacteria in Clear and Humic Waters

In highly humic water, acridine orange precipitated with dissolved humic matter, resulting in such bright background fluorescence that no bacteria could be seen. With bisbenzimide staining, a similar precipitate was nonfluorescent but obscured many cells. An acriflavine staining method proved useful and reproducible both in clear and in humic waters. Fading of fluorescence was not a problem, and stained samples could be stored after preparation. The fluorescence of cells stained with acriflavine was weaker than that with acridine orange, making counting extremely small cells slightly more difficult with the former stain.     

Evidence for the contribution of humic substances to conditioning films from natural waters

The presence of humic substances in conditioning films deposited on solid surfaces from natural waters was investigated using electron impact (EI), chemical ionization (CI) and secondary ion time-of-flight mass spectrometry (TOFSIMS). EI and CI spectra of a freshwater sample from a pond in Centennial Park, Sydney, Australia, showed a high degree of similarity with spectra of humic acids purchased from Fluka and Sigma as well as with reference humic acid and fulvic acid from the International Humic Substances Society, suggesting that most of the organic matter in the pond water was of humic origin. All the complex electron impact mass spectra feature series of high-intensity ions separated by 14 Da or 18 Da, which can be attributed to CH(2) and OH(2) respectively. Thermal desorption profiles of all samples generated by EI and CI were qualitatively similar. The secondary desorption peaks were less well-defined in CI compared to EI. Positive ion thermal desorption profiles displayed a two-step ionisation, with a sharp and well-defined initial desorption peak at t～50s, followed by a broader desorption peak with a maximum intensity at t ～ 100 s post-heating. The Centennial Park natural organic matter (NOM) differed from the other humic fractions in having two additional broad desorption peaks between the two described previously, and a less-defined initial peak. Infrared spectroscopy showed that proteinaceous matter in the lake water was insignificant in comparison with functional groups indicative of humic substances. TOFSIMS characterization showed almost identical spectra for Aldrich humic acid and Centennial Park NOM in the high mass region of 2000 Da to 3000 Da. Each spectrum contains approximately 25 groups of ion peaks, separated by 74 Da from group to group. Each group is composed of 6 or 7 individual peaks. The spectral features are consistent with a macromolecular structure of humic acid where aromatic rings are joined to the macrostructure via aliphatic spacer molecules.     

Microbial transformation of labile dissolved organic matter into humic-like matter in seawater

Abstract Microbial transformation of labile, low molecular weight dissolved organic matter (DOM) into dissolved humic matter (DHM) was studied in seawater. Surface water samples were amended with [¹⁴C into ¹⁴CO₂, TO¹⁴C (total organic ¹⁴C), and PO¹⁴C (particulate organic ¹⁴C), was measured over time in confined samples. The humic and non-humic fractions of DO¹⁴C (dissolved organic ¹⁴C) were separated according to a common operational definition of DHM based on adsorption on XAD-8 macroporous resin. Both TO¹⁴C and non-humic DO¹⁴C decreased during the experiments. However, ¹⁴C-labelled DHM increased during the first week of the incubations, to a level where it comprised 15% of the TO¹⁴C remaining in the samples, or 3% of the initially added ¹⁴C. Towards the end of experiments (ca 70 days), the humic fraction of DO¹⁴C gradually approached the background level of poisoned control samples. Provided that the XAD-8 operational definition of DHM is accepted, this study indicates that humic matter may be formed in seawater within days from labile monomers such as glucose.     

The Influence of Organic Matter and pH on DDT Aqueous Solubility

We have studied the concentrations of DDT in ground water samples at field locations with DDT-polluted topsoil and concentrations and solubility in samples prepared from deionized water with different types and concentration of organic acids. The solubility of DDT increased with increasing concentration of humic acid when the pH of the samples was low (adjusted to about 5.5). The effect flutters in the humic acid concentration range from 200 to 300?mg/L, in accordance with humic acid hydrophobicity, operationally measured as liquid surface tension. The findings correspond to trends previously reported in the literature. The trend of increasing solubility was not found using fulvic acid or low-molecular-weight aliphatic acids. No trend was found adding humic acid without adjusting the pH. The mechanism of enhanced solubility due to humic compounds can explain relatively high levels of DDT in ground water. The ground water samples, however, had a moderately high concentration of maximum 6?µg/L compared with a maximum of about 2300?µg/L in the water samples with humic acid in pure water.     

Relationships between mercury in lake water,water colour and mercury in fish

The relationship between mercury content in fish (pike and perch), the different fractions of mercury in lake water and water color was investigated in 76, mainly oligotrophic lakes distributed over a large part of Sweden. The lakes were classified in terms of drainage area characteristics, lake morphometry and water chemistry. The dominant fraction of mercury in lake water was RIHg (fraction reducible to elemental mercury by NaBH₄). RIHg and water color were strongly positively correlated. Water color (determined by the comparative method using colored disks) was used as a surrogate for the amount of humic matter in the water. Thus, humic matter appears to be acting as an important carrier of mercury. A positive relationship between mercury content in fish and water color was found only in deep lakes (average depth > 5 m). It is suggested that the bioavailability of mercury attached to humic matter increases due to anoxic conditions, common in the hypolimnion of deep lakes.     

Application of alkaline treatment for sludge decrement and humic acid recovery

A new method was introduced to reduce waste activated sludge and extract humic acid for liquid fertilizer. Sludge was disintegrated with NaOH (0.4 g/g dry solid, 8 h) and then centrifuged to obtain the supernatant. The residual sludge was then dewatered, while the supernatant was used to extract humic acid with an ultrafiltration membrane. The results showed that the alkaline treatment dissolved more than half of the sludge organic matter, which was composed of 24% humic acid by mass. After the supernatant was concentrated 20 times using a membrane with a molecular weight cutoff of 1000, the retentate contained 94.5% of the dissolved organics and could be used to produce humic acid fertilizer. Additionally, only 26% of the NaOH was consumed and the residual NaOH in the permeate flux could be reused. Due to the removal of water and organics, the dewatered sludge could be reduced by 60% when compared to samples that did not receive the alkaline treatment.     

Effects of zooplankton carcasses degradation on freshwater bacterial community composition and implications for carbon cycling

Non-predatory mortality of zooplankton provides an abundant, yet, little studied source of high quality labile organic matter (LOM) in aquatic ecosystems. Using laboratory microcosms, we followed the decomposition of organic carbon of fresh ¹³C-labelled Daphnia carcasses by natural bacterioplankton. The experimental setup comprised blank microcosms, that is, artificial lake water without any organic matter additions (B), and microcosms either amended with natural humic matter (H), fresh Daphnia carcasses (D) or both, that is, humic matter and Daphnia carcasses (HD). Most of the carcass carbon was consumed and respired by the bacterial community within 15 days of incubation. A shift in the bacterial community composition shaped by labile carcass carbon and by humic matter was observed. Nevertheless, we did not observe a quantitative change in humic matter degradation by heterotrophic bacteria in the presence of LOM derived from carcasses. However, carcasses were the main factor driving the bacterial community composition suggesting that the presence of large quantities of dead zooplankton might affect the carbon cycling in aquatic ecosystems. Our results imply that organic matter derived from zooplankton carcasses is efficiently remineralized by a highly specific bacterial community, but does not interfere with the bacterial turnover of more refractory humic matter.     

Sources,concentrations and characteristics of organic matter in softwater lakes and streams of the Swedish forest region

18 Swedish forest lakes covering a wide range of dystrophy were studied in order to quantify and characterize the organic matter in the water with respect to origin (allochthonous or autochthonous), physical state (particulate or dissolved) and phosphorus content. Samples were collected repeatedly during a two-year period with unusually variable hydrological conditions. Water from three different depths and from tributaries was analysed with standard monitoring methods, including water colour, Secchi disk transparency, total organic carbon (TOC), COD_Cr, COD_Mn, total phosphorus and molybdate reactive phosphorus. Interrelationships were used to compare different methods and to assess the concentration and composition of organic matter. It is estimated that in remote softwater lakes of the Swedish forest region, autochthonous carbon is typically < 5 g m⁻³. Most lakes in this region receive significant amounts of humic matter originating from coniferous forest soils or peatland in the catchment area. In most humic lakes with a water colour of ≥ 50 g Pt m⁻³, more than half of the organic carbon in the surface water is of allochthonous origin, and in polyhumic lakes (> 200 g Pt m⁻³) the proportion can exceed 90%. Secchi depth readings were related similarly to organic matter from both sources and provided good estimates of TOC with a single optical measurement. Water colour was used to distinguish allochthonous and autochthonous matter. High concentrations of phosphorus were found in humic waters, most of it being molybdate reactive, and probably associated with humic matter rather than as dissolved free inorganic forms. COD_Mn yielded only 25–60% of TOC and appears to include mainly truly dissolved substances of low molecular weight.     

How light and nutrients affect the relationship between autotrophic and heterotrophic biomass in a tropical black water periphyton community

This study examines how nutrients and light affect the relationship between autotrophic biomass and non-autotrophic periphyton organic matter in a tropical black water lake biofilm community. We hypothesized that there is no positive correlation between autotrophic and non-autotrophic organic matter in the periphytic community of a black water humic lake, where non-algal components of periphyton can rely on carbon sources external to the periphyton matrix and where nutrient availability is low. Second, we sought to test our hypothesis that non-autotrophic periphyton organic matter will benefit from nutrient enhancement in a lake where the availability of DOC is high. We performed a field experiment using in situ lake mesocosms to manipulate nutrient concentrations and light availability in a 2 × 2 factorial design. Control treatments (no nutrient added) and nutrient treatments (N + P) were compared in different light conditions: high light (near surface water) and low light (near bottom). No positive correlation was found between autotrophic biomass and non-autotrophic periphyton organic matter, but a negative correlation was observed in high nutrient and light conditions. The low C:P and N:P ratios revealed that the non-autotrophic organic matter mostly comprised a heterotrophic microbial biofilm. High levels of light and nutrients together caused significant changes in periphyton community properties. The non-autotrophic periphyton organic matter was negatively affected by nutrient addition, whereas autotrophic biomass was positively affected, especially in high light conditions. Our results strongly suggest that non-autotrophic periphyton organic matter in a humic lake is primarily comprised of a bacterial biofilm that directly competes for nutrients with autotrophs in the periphytic community. We also observed no effect of nutrient addition on periphyton growing in light-limited conditions. These results suggest that heterotrophic periphytic organisms might experience carbon limitation despite the high availability, but usually low quality, of dissolved carbon in the water column of humic lakes.     

Pyrolysis-field ionization mass spectrometry of agricultural soils and humic substances: Effect of cropping systems and influence of the mineral matrix

Whole soil samples, extracted humic substances, the corresponding fulvic (FA) and humic acids (HA) and the extraction residues (humins) from long-term, agricultural test plots were investigated by in-source pyrolysis-field ionization mass spectrometry (Py-FIMS). For the soils distinct differences in the chemical composition of the organic matter in differently managed fields were observed. The FI mass spectra of the extracted humic substances gave complementary chemical information, as they cover a larger mass range compared to the whole soil spectra. The chemical, structural information of the conventional alkaline extraction residues was demonstrated by Py-FIMS spectra to be similar to that of the related soil samples. Influences of mineral matrix to organic matter ratios were studied on mixtures of extracted humic substances with defined mineral components such as quartz, basalt, iron oxide (Fe₂O₃), Ca-montmorillonite, kaolinite and illite. It was shown that in these mixtures the number of mass signals detected and the covered mass range decreased, when organic carbon concentrations (C_org) in this synthetic mineral matrix dropped below 2% (w/w). Limitations in the direct application of Py-FIMS might arise in the case of natural soil samples with C_org concentrations below 0.5% (w/w), high contents of swelling clay minerals and iron oxides. ei]{gnR}{fnMerckx}     

Effects of the Addition of Humic Substances on Soil Protease Activities in Andosols in the Presence of Cadmium

Cadmium extraction, sorption, and immobilization seem to be the effective mechanisms in detoxification of Cd-contaminated soil. Humic substances present in soils may play an important role both in controlling the negative effects of pollution with Cd and in stabilizing soil enzymes. In this context, we have compared the effects of humic substances on soil protease activities in the presence and absence of Cd in forest and cultivated field soil samples. The samples were taken from surface soils of Andosols in a single upland area of the Kanto district in Japan. Humic substances extracted from the two soils showed differences in elemental composition, the degree of condensation of aromatic groups, and the proportions of major functional groups. Compared with the control soil samples, those with added humic substances showed a significant increase in protease activities, even in the presence of Cd (10 and 50 mg Cd kg^?1 soil). However, the addition of Cd decreased the protease activities, protein contents, and soil pH in both soil samples at each of the two levels of humic substance fortification (+5% and +10%). Moreover, protease activities showed significant negative correlation with exchangeable Cd, but adding humic substances did not lead to a reduction in either sample. Thus, although the addition of humic substances increased and stabilized protease activities, it did not lead to a clear reversal of enzyme inhibition by Cd. The obtained results indicate that in both soil samples the humic substances used in this study did not have sufficient affinity to adsorb Cd, and the impact on enzyme activities depends on the difference in chemical characteristics of the added organic matter, as suggested by the difference in enhancement of protease activities between forest and cultivated field soil samples.     

Use of measurement of liver cytochrome P 450 as a water toxicity test]

The authors describe a new approach to determine the toxicity of water samples. The level of liver hydroxylating cytochrome in mice injected with water samples is assayed. This assay is used to test the toxicity of waters containing an earth extract (humic and fulvic acids). The water samples showed various toxicities desinfected with chlorine or ozone.     

Seasonal changes in the concentration and colour of humic substances in some aquatic environments

SUMMARY. 1. Between February and September 1974, concentrations of fulvic and humic acids in waters from a cryoboreal region of the Precambrian Shield in Quebec varied between 0.5–6.1 mg 1⁻¹ and 0.1–6.5 mg 1⁻¹ respectively. Whereas the fulvic acids usually remained relatively constant during this period, humic acids showed appreciable increases.
2. The number and weight average molecular weights of the fulvic acids (∼5000 and ∼22,000 daltons respectively) were lower than those of the humic acids (∼20,000 and ∼75,000 daltons respectively). The average molecular weight of the aquatic humic matter often decreased during late summer and early autumn. During spring floods the molecular weight of fulvic acids increased and that of humic acids declined.
3. Because the aquatic humic acids were more intensely coloured than fulvic acids, they contributed more than 30% of the colour of the water even though they accounted for only 15% of the concentration of humic matter. The colour intensity of both fulvic and humic acids generally appeared to increase during the latter part of the summer, when there was normally a decrease in the value of the colour quotient (E₄/E₆ ratio), indicating the accumulation in the waters of more humified material.     

Interactions between humic matter and bacteria when disinfecting water with UV light

Aims:  To investigate the impact of aquatic humic matter on the inactivation of Escherichia coli and Bacillus subtilis by ultraviolet (UV) light.
Methods and Results:  A bench-scale study investigated the potential for Aldrich^® humic acid (AHA) and Suwannee River natural organic matter (SR-NOM) to coat the surface of E. coli and B. subtilis and offer protection from low-pressure UV light. UV doses of 5 and 14 mJ cm⁻² were applied using a collimated beam at four concentrations of humic matter (0, 10, 50 and 120 mg l⁻¹) in reagent grade water. Both AHA and SR-NOM were found to offer statistically significant protection of both E. coli and B. subtilis at concentrations of 50 and 120 mg l⁻¹ for a UV dose of 14 mJ cm⁻².
Conclusions:  Both E. coli and B. subtilis are susceptible to coating by humic matter which can reduce the sensitivity of the cells to UV light.
Significance and impact of the study:  Micro-organisms in the environment may acquire characteristics through interaction with humic matter that render them more resistant to UV disinfection than would be predicted based on laboratory inactivation studies using clean cells.     

Littoral-pelagial interchange and the decomposition of dissolved organic matter in a polyhumic lake

The small, polyhumic lake, Mekkojärvi (southern Finland), is bordered by a moss vegetation zone (Warnstorfia and Sphagnum species) which provides a habitat-rich and productive environment for many planktonic and periphytic animals. Impacts of moss on the metabolism of bacterioplankton, phytoplankton and zooplankton in polyhumic water were investigated in laboratory throughflow systems. Growing Warnstorfia (together with epiphytic algae and bacteria) suppressed the production of planktonic algae but had no clear effect on leucine uptake, and hence bacterial production, or on the decomposition of humic substances. Phenol uptake and mineralization rates, however, were lower in the littoral water than in the pelagial water. Excretion of organic carbon by Warnstorfia algae or Daphnia longispina (the predominant crustacean in the pelagial water) provided only a minor contribution to bacterial production; therefore, a major contribution had to be from humic substances. A bacterial production efficiency of 31–38% could account for the microbial respiration in the water. The results indicated that bacterial, or detrital matter (originating largely from the littoral zone), could not obviate the need for algal food, and that a great deal of particulate matter in the water was poor or useless food for Daphnia. In all, the bulk of dissolved organic matter in Lake Mekkojärvi was biochemically highly recalcitrant. Our results indicate that humic substances (from watershed or littoral area) which, through bacterial degradation, enter the planktonic food web of the lake are mainly lost through respiration by microorganisms.     

The disruption of Daphnia magna sodium metabolism by humic substances: mechanism of action and effect of humic substance source

Humic substances have important functions in aquatic systems. While these roles are primarily indirect, influencing the physicochemical environment, recent evidence suggests these materials may also have direct biological actions. This study investigated the mechanism by which humic substances perturb sodium metabolism in a freshwater invertebrate, the water flea Daphnia magna. Aldrich humic acid (AHA) stimulated the maximal rate of whole-body sodium influx (Jmax) when experimental pH was 6 and water calcium content was 0.5 mM. This effect persisted at pH 8 and 1 mM calcium but not at pH 8 in the absence of calcium. An indirect action of AHA on apical transporter activity was proposed to explain this effect. At pH 4 AHA promoted a linear sodium uptake kinetic relationship, attributed to altered membrane permeability due to enhanced membrane binding of humic substances at low pH. In contrast, a real-world natural organic matter sample had no consistent action on sodium influx, suggesting that impacts on sodium metabolism may be limited to commercially available humic materials. These findings question the applicability of commercially available humic substances for laboratory investigations and have significant implications for the study of environmental metal toxicity.     

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.     

On the importance of dissolved organic matter in the nutrition of zooplankton in some lake waters

Summary A steady state, radiotracer technique was used to study the original source of the carbon in zooplankton. The experiments were started in filtered lake water with added inorganic radiocarbon. At the beginning of the experiments, a proportionally insignificant volume of unfiltered water was introduced into the culture, together with some ovigerous zooplankton individuals. Since the radioactivity: carbon ratio in the dissolved inorganic carbon was kept constant, a similar ratio would be expected to develop in the autotrophic phytoplankton. The same ratio would then be expected to develop in the zooplankton, if its sole carbon source was autotrophic phytoplankton.According to the results of this approach dissolved organic matter seems to be an important food resource for zooplankton, particularly in highly humic lakes. This conclusion was confirmed by the finding that zooplankton from these lakes was able to grow and reproduce in experiments started with filtered lake water and conducted in complete darkness.The development of algae was followed over the course of one experiment in highly humic water. The same micro-flagellates reproduced equally well in both light and darkness, which indicates the importance of heterotrophic metabolism in their nutrition. Although there are no direct observations about the food of zooplankton in our experiments, it appears likely that heterotrophic flagellates play an important role as a food of zooplankton in humic waters.The importance of dissolved organic matter in the nutrition of aquatic organisms would seem to be much greater than has generally been recognized. Consequently the prevailing concepts of the structure and functioning of planktonic ecosystem should be thoroughly re-evaluated.     

Effect of humic substance photodegradation on bacterial growth and respiration in lake water

This study addresses how humic substance (HS) chemical composition and photoreactivity affect bacterial growth, respiration, and growth efficiency (BGE) in lake water. Aqueous solutions of HSs from diverse aquatic environments representing different dissolved organic matter sources (autochthonous and allochthonous) were exposed to artificial solar UV radiation. These solutions were added to lake water passed through a 0.7-microm-pore-size filter (containing grazer-free lake bacteria) followed by dark incubation for 5, 43, and 65 h. For the 5-h incubation, several irradiated HSs inhibited bacterial carbon production (BCP) and this inhibition was highly correlated with H2O2 photoproduction. The H2O2 decayed in the dark, and after 43 h, nearly all irradiated HSs enhanced BCP (average 39% increase relative to nonirradiated controls, standard error = 7.5%, n = 16). UV exposure of HSs also increased bacterial respiration (by approximately 18%, standard error = 5%, n = 4), but less than BCP, resulting in an average increase in BGE of 32% (standard error = 10%, n = 4). Photoenhancement of BCP did not correlate to HS bulk properties (i.e., elemental and chemical composition). However, when the photoenhancement of BCP was normalized to absorbance, several trends with HS origin and extraction method emerged. Absorbance-normalized hydrophilic acid and humic acid samples showed greater enhancement of BCP than hydrophobic acid and fulvic acid samples. Furthermore, absorbance-normalized autochthonous samples showed approximately 10-fold greater enhancement of BCP than allochthonous-dominated samples, indicating that the former are more efficient photoproducers of biological substrates.     

Effects of humic substances on fluorometric DNA quantification and DNA hybridization

DNA extracts from sediment and water samples are often contaminated with coextracted humic-like impurities. Estuarine humic substances and vascular plant extract were used to evaluate the effect of the presence of such impurities on DNA hybridization and quantification. The presence of humic substances and vascular plant extract interfered with the fluorometric measurement of DNA concentration using Hoechst dye H33258 and PicoGreen reagent. Quantification of DNA amended with humic substances (20-80 ng/microl) using the Hoechst dye assay was more reliable than with PicoGreen reagent. A simple procedure was developed to improve the accuracy for determining the DNA concentration in the presence of humic substances. In samples containing up to 80 ng/microl of humic acids, the fluorescence of the samples were measured twice: first without Hoechst dye to ascertain any fluorescence from impurities in the DNA sample, followed with Hoechst dye addition to obtain the total sample fluorescence. The fluorescence of the Hoechst dye-DNA complex was calculated by subtracting the fluorescence of the impurities from the fluorescence of the sample. Vascular plant extract and humic substances reduced the binding of DNA onto the nylon membrane. Low amounts (<2.0 microg) of humic substances derived from estuarine waters did not affect the binding of 100 ng of target DNA to nylon membranes. DNA samples containing 1.0 microg of humic substances performed well in DNA hybridizations with DIG-labeled oliogonucleotide and chromosomal probes. Therefore, we suggest that DNA samples containing low concentrations of humic substances (<20 ng/microl) could be used in quantitative membrane hybridization without further purification.