Assessment of artificial substrates for evaluating groundwater microbial quality

Protection of groundwater resources requires the development of reliable ecological indicators. Microorganisms involved in ecological services or being associated with particular hosts or habitats could be used for this purpose. Nevertheless, their tracking remains limited because of sampling issues, and a lack of devices for their long term monitoring. In the present study, three artificial substrates (glass and clay beads, and gravel particles) were tested in terms of efficacy at favoring bacterial growth, and at capturing bacterial diversity of waters (i.e., groundwater, surface water and wastewater). Total proteins, total carbohydrates, dehydrogenase and hydrolytic activities were used to monitor biofilm development on these artificial substrates. Fingerprinting analyses based on rrs (16S rRNA) − rrl (23S rRNA) spacer analyses (ARISA) and next generation sequencing (NGS) of partial rrs DNA segments (V5-V6) were used to compare operating taxonomic units (OTUs), and infer bacterial genera trapped on these substrates. Glass beads were found less efficient than the other two artificial substrates at increasing protein contents and microbial activities (hydrolytic and dehydrogenase activities). ARISA showed a discrimination of bacterial communities developing on artificial substrates that was matching water types. An incubation period of 7 days allowed a reliable assessment of bacterial diversity. From this incubation period, around 75% of water genera with more than four V5-V6 rrs DNA sequences detected in a water type were recovered from biofilms growing on artificial substrates. Based on relative abundances of genera, clay beads and gravel particles were more efficient than glass beads to capture and obtain bacterial communities matching those of the initial waters. Between 45–67% of similarities were found for these artificial substrates while it was between 36 and 43% for glass beads. This study demonstrated clay beads and gravel particles as being efficient tools for capturing bacterial diversity and monitoring bacterial growth. Overall, clay beads appeared the best choice for field monitoring because of the ease of their size standardization in comparison with gravel particles.     

Surface Water Linkages Regulate Trophic Interactions in a Groundwater Food Web

Groundwaters are increasingly viewed as resource-limited ecosystems in which fluxes of dissolved organic carbon (DOC) from surface water are efficiently mineralized by a consortium of microorganisms which are grazed by invertebrates. We tested for the effect of groundwater recharge on resource supply and trophic interactions by measuring physico-chemistry, microbial activity and biomass, structure of bacterial communities and invertebrate density at three sites intensively recharged with surface water. Comparison of measurements made in recharge and control well clusters at each site showed that groundwater recharge significantly increased fluxes of DOC and phosphate, elevated groundwater temperature, and diminished dissolved oxygen (DO). Microbial biomass and activity were significantly higher in recharge well clusters but stimulation of autochthonous microorganisms was not associated with a major shift in bacterial community structure. Invertebrate assemblages were not significantly more abundant in recharge well clusters and did not show any relationship with microbial biomass and activity. Microbial communities were bottom-up regulated by DOC and nutrient fluxes but trophic interactions between microorganisms and invertebrates were apparently limited by environmental stresses, particularly DO depletion and groundwater warming. Hydrological connectivity is a key factor regulating the function of DOC-based groundwater food webs as it influences both resource availability for microorganisms and environmental stresses which affect energy transfer to invertebrates and top-down control on microorganisms.     

Spatio-temporal patterns of microbial communities in a hydrologically dynamic pristine aquifer

Seasonal patterns of groundwater and sediment microbial communities were explored in a hydrologically dynamic alpine oligotrophic porous aquifer, characterized by pronounced groundwater table fluctuations. Rising of the groundwater level in consequence of snow melting water recharge was accompanied by a dramatic drop of bacterial Shannon diversity in groundwater from H' = 3.22 ± 0.28 in autumn and winter to H' = 1.31 ± 0.35 in spring and summer, evaluated based on T-RFLP community fingerprinting. Elevated numbers of bacteria in groundwater in autumn followed nutrient inputs via recharge from summer rains and correlated well with highest concentrations of assimilable organic carbon. Sterile sediments incubated to groundwater in monitoring wells were readily colonized reaching maximum cell densities within 2 months, followed by a consecutive but delayed increase and leveling-off of bacterial diversity. After 1 year of incubation, the initially sterile sediments exhibited a similar number of bacteria and Shannon diversity when compared to vital sediment from a nearby river incubated in parallel. The river bed sediment microbial communities hardly changed in composition, diversity, and cell numbers during 1 year of exposure to groundwater. Summing up, the seasonal hydrological dynamics were found to induce considerable dynamics of microbial communities suspended in groundwater, while sediment communities seem unaffected and stable in terms of biomass and diversity.     

Influences of the petroleum-recovering activity on the arsenic level in groundwater of Kuitun,Xinjiang, China

Increasing demands of groundwater in petroleum-recovering regions could elevate the level and mobility of arsenic in groundwater as a result of the enhanced dissolution of arsenic-bearing iron or manganese oxide due to the accelerated sulfate reduction by microorganisms in a reductive environment. To substantiate this possibility, groundwater samples were collected from 220 water wells in the nearby petroleum wells in Kuitun. Dissolved arsenic, iron, manganese, and sulfate levels and pH in groundwater samples were analyzed. The dissolved arsenic levels in groundwater varied from <2.3 to 789.4 μg·L^?1, in which approximately 96.4% of the measured values exceeded the allowed limits of the World Health Organization. An inverse relation existed between dissolved arsenic and sulfate levels. Most of the high arsenic-level samples (>300 μg·L^?1) were found in wells at close proximity to petroleum wells where a high iron or manganese level was also detected. The oil-exploring activity in the study region seemed to have enhanced the microbial reduction of sulfate in underground environment and hence the level of arsenic in groundwater. The microbial sulfate reduction coupled with the reduction of arsenic-bearing iron oxides in the groundwater environment may explain the spatial heterogeneity of the arsenic level in groundwater.     

Bioreduction of U(VI) in groundwater under anoxic conditions from a decommissioned in situ leaching uranium mine

To determine whether the U(VI) in groundwater under anoxic conditions at a decommissioned in situ leaching (ISL) uranium mine could be bioreduced, groundwater samples containing suspended sediments were taken from the mine, experimental setup was fabricated, and the jar containing the groundwater in the setup was amended with ethanol and incubated under anoxic conditions. The variations of pH, chemical oxygen demand, nitrate, sulfate, U(VI), and dissolved oxygen (DO) concentrations were monitored during the incubation. U(VI) concentration dropped to 0.043 mg/L when the stimulated microorganisms were active, and it then increased to 0.835 mg/L within 10 days after the metabolism of the stimulated microorganisms was inhibited. The DO variation was observed in the amended jar during the incubation, and the metabolism of the stimulated microorganisms was found to affect the DO concentration. Firmicutes were found to be dominant in the sediments in the amended jar through the 16S rRNA pyrosequencing. The results indicate that it is possible to bioreduce U(VI) in the groundwater under anoxic conditions at the decommissioned ISL uranium mine by adding carbon source into it without removing the oxygen from it.     

Utilization of Microbial Biofilms as Monitors of Bioremediation

A down-well aquifer microbial sampling system was developed using glass wool or Bio-Sep beads as a solid-phase support matrix. Here we describe the use of these devices to monitor the groundwater microbial community dynamics during field bioremediation experiments at the U.S. Department of Energy Natural and Accelerated Bioremediation Research Programs Field Research Center at the Oak Ridge National Laboratory. During the 6-week deployment, microbial biofilms colonized glass wool and bead internal surfaces. Changes in viable biomass, community composition, metabolic status, and respiratory state were reflected in sampler composition, type of donor, and groundwater pH. Biofilms that formed on Bio-Sep beads had 2–13 times greater viable biomass; however, the bead communities were less metabolically active [higher cyclopropane/monoenoic phospholipid fatty acid (PLFA) ratios] and had a lower aerobic respiratory state (lower total respiratory quinone/PLFA ratio and ubiquinone/menaquinone ratio) than the biofilms formed on glass wool. Anaerobic growth in these systems was characterized by plasmalogen phospholipids and was greater in the wells that received electron donor additions. Partial 16S rDNA sequences indicated that Geobacter and nitrate-reducing organisms were induced by the acetate, ethanol, or glucose additions. DNA and lipid biomarkers were extracted and recovered without the complications that commonly plague sediment samples due to the presence of clay or dissolved organic matter. Although microbial community composition in the groundwater or adjacent sediments may differ from those formed on down-well biofilm samplers, the metabolic activity responses of the biofilms to modifications in groundwater geochemistry record the responses of the microbial community to biostimulation while providing integrative sampling and ease of recovery for biomarker analysis. Dedication: The authors dedicate this paper to Professor Peter Hirsch, whose infectious enthusiasm for beautiful mushrooms, Antarctica, and most of all weird microbes he has isolated and characterized has greatly enriched our lives as microbiologists with the wonder of what lies beyond our lab-rat cultures. Once Peter visits your lab, looking through your microscope will be a different experience. Happy birthday and thanks for so enriching our lives and profession.     

Enhanced accumulation of starch and total carbohydrates in alginate-immobilized Chlorella spp. induced by Azospirillum brasilense: I. Autotrophic conditions

The effect of the microalgae-growth promoting bacterium Azospirillum brasilense on accumulation of total carbohydrates and starch in two species of Chlorella (Chlorella vulgaris and Chlorella sorokiniana), when the bacterium and each microalga were jointly immobilized in alginate beads was studied under autotrophic conditions for 144h in synthetic medium. The interaction of the bacterium with the microalgae enhanced accumulation of total carbohydrate and starch. Cells of Chlorella accumulated the highest amounts of carbohydrate after incubation for 24h. Yet, this did not coincide with the highest affinity and volumetric productivity measured in these cultures. However, after incubation for 72h, mainly in jointly immobilized treatments of both microalgae species, the cultures reached their highest total carbohydrate content (mainly as starch) and also the highest affinity and volumetric productivity. These results demonstrate the potential of A. brasilense to affect carbohydrates and starch accumulation in Chlorella spp. when both microorganisms are co-cultured, which can be an important tool for applications of microalgae.     

Vertical change in dissolved organic carbon and oxygen at the water table region of an aquifer recharged with stormwater: biological uptake or mixing?

Decreases in dissolved organic carbon (DOC) and dissolved oxygen (DO) with increasing depth below the groundwater table are often considered as evidence for aerobic respiration; however, they may reflect mixing of infiltrating water and groundwater. We found that groundwater DOC concentration was on average 0.3 mg C l^?1 higher and DO concentration 1.5 mg O₂ l^?1 lower at recharge sites replenished with stormwater than at reference sites fed by direct infiltration of rain water from the land surface. Groundwater DOC increased and DO decreased with increasing vadose zone thickness (VZT) at both recharge and reference sites. There was no significant interaction between the effects of stormwater infiltration and VZT. Vertical changes in DOC and DO below the groundwater table at recharge sites could account for by simple mixing of infiltrating stormwater and groundwater. Moreover, aquifer sediment respiration (SR) was not significantly higher at recharge sites than at reference sites. However, slow filtration column experiments showed that SR increased significantly with an increasing supply of easily biodegradable DOC. We conclude that the observed reduction in DOC below the groundwater table at recharge sites was essentially due to water mixing rather than biological uptake because of the low biodegradability of the DOC and the short transit time of stormwater in the upper layers of groundwater. Our results highlight the need to distinguish between the effect of hydrological and biological processes on DOC and DO patterns below the groundwater before conclusions are made on the efficiency of groundwater in degrading surface-derived DOC.     

The impact of dissolved amino acids on protein and cellulose degradation in stream waters

Dissolved amino acids represent a significant carbon and nitrogen source for microorganisms in stream water environments, and may be utilized in preference to protein bound nitrogen in decomposing organic matter. Consequently, in streams with sufficiently high concentrations of dissolved amino acids, depolymerisation of nitrogen compounds may become delayed. This possibility was investigated in stream water samples incubated with ¹⁴C-labelled albumin and cellulose in presence of the indigenous microorganisms at different concentrations of dissolved amino acids.The experiments demonstrated at an average between 15 and 25% lower degradation of the compounds during a ten days incubation at an initial amino acid concentration of 1 mg l^–1. The reduction was most clearly expressed in a nutrient-poor stream water compared with a nutrient-rich. The kinetics of the degradation were most appropriately described by a first-order model, that is, the rate of transformation of the macromolecules was independent of the total number of bacteria in the water. The mechanism suggested for the retardation of macromolecules is a superproportional utilization of the dissolved amino acids at high concentrations, a phenomenon that can cause accumulation of slowly decomposing macromolecules in sediments affected by residual wastewater.     

Removal of gaseous n-valeric acid in the air by Rhodococcus sp. B261 immobilized onto ceramic beads

n-Valeric acid, one of the main malodorous pollutants from livestock houses was eliminated with a biofilter prepared with Rhodococcus sp. B261 immobilized onto ceramic beads. The strain was isolated from composted pig faeces and grown in an artificial medium containing volatile fatty acids as a carbon source. The cells were immobilized onto ceramic beads in vacuo. The beads were aseptically incubated at 37 °C, pH 8.0, for 24h for activation of the cells. The beads with immobilized cells (3.36×10⁹ c.f.u./g ceramic beads) and moisture content of 35% (w/w) were packed into a glass column equipped with a water jacket to keep the temperature constant. One hundred-seventy ppm of gaseous n-valeric acid were removed for 11 days at 30h ^-1 (space velocity) and 37 °C.     

Microbiological Comparison of Core and Groundwater Samples Collected from a Fractured Basalt Aquifer with that of Dialysis Chambers Incubated In Situ

Microorganisms associated with basalt core were compared to those suspended in groundwater pumped from the same well in the eastern Snake River Plain Aquifer (Idaho, USA). Two wells located at different distances from the source of a mixed-waste plume in the fractured basalt aquifer were examined. In the well more distal from the plume source, an array of dialysis chambers filled with either deionized water or crushed basalt was equilibrated to compare the microorganisms collected in this fashion with those from core and groundwater samples collected in a traditional manner from the same well. The samples were characterized to determine the total amount of biomass, presence of specific populations or physiological groups, and potential community functions. Microorganisms and their activities were nearly undetectable in core and groundwater collected from the well farthest from the plume source and substantially enriched in both core and groundwater from the well closest to the plume source. In both wells, differences (statistically significant for some measures) were found between bacteria associated with the cores and those suspended in the groundwater. Significantly higher populations were found in the basalt- and water-filled dialysis chambers incubated in the open well compared with core and groundwater samples, respectively. For a given parameter, the variation among dialysis chambers incubated at different depths was much less than the high variation observed among core samples. Analyses on selected basalt- and water-filled dialysis chamber samples suggested that these two communities were compositionally similar but exhibited different potential functions. Documented knowledge of cell physiological changes associated with attachment and potential differences between attached and unattached communities in aquifers indicate that careful consideration should be given to the type of sample media (i.e., core, groundwater, substrata incubated in a well) used to represent a subsurface environment.     

Biogas production using anaerobic groundwater containing a subterranean microbial community associated with the accretionary prism

In a deep aquifer associated with an accretionary prism, significant methane (CH₄) is produced by a subterranean microbial community. Here, we developed bioreactors for producing CH₄ and hydrogen (H₂) using anaerobic groundwater collected from the deep aquifer. To generate CH₄, the anaerobic groundwater amended with organic substrates was incubated in the bioreactor. At first, H₂ was detected and accumulated in the gas phase of the bioreactor. After the H₂ decreased, rapid CH₄ production was observed. Phylogenetic analysis targeting 16S rRNA genes revealed that the H₂-producing fermentative bacterium and hydrogenotrophic methanogen were predominant in the reactor. The results suggested that syntrophic biodegradation of organic substrates by the H₂-producing fermentative bacterium and the hydrogenotrophic methanogen contributed to the CH₄ production. For H₂ production, the anaerobic groundwater, amended with organic substrates and an inhibitor of methanogens (2-bromoethanesulfonate), was incubated in a bioreactor. After incubation for 24 h, H₂ was detected from the gas phase of the bioreactor and accumulated. Bacterial 16S rRNA gene analysis suggested the dominance of the H₂-producing fermentative bacterium in the reactor. Our study demonstrated a simple and rapid CH₄ and H₂ production utilizing anaerobic groundwater containing an active subterranean microbial community.     

Increase of the activity state and loss of total activity of the branched-chain 2-oxo acid dehydrogenase in rat diaphragm during incubation.

Actual and total branched-chain 2-oxo acid dehydrogenase activities were determined in homogenates of incubated diaphragms from fed and starved rats. Incubation in Krebs-Ringer buffer increased the activity state, but caused considerable loss of total activity. Palmitate oxidation rates and citrate synthase activities did not significantly change on incubation. Starved muscles showed a higher extent of activation after 15 min of incubation (not after 30 and 60 min) and a smaller loss of total activity. Experiments with the transaminase inhibitor amino-oxyacetate confirm that the contribution of endogenous amino acids to the oxidation precursor pool is also smaller in diaphragms from starved rats on incubation in vitro. These phenomena together cause the higher 14CO2 production from 14C-labelled branched-chain amino acids and 2-oxo acids in muscles from starved than from fed rats. High concentrations of branched-chain 2-oxo acids, and the presence of 2-chloro-4-methyl-pentanoate, octanoate or ketone bodies, increase the extent of activation of the dehydrogenase complex; glucose and pyruvate had no effect. The observed changes of the activity state by these metabolites are discussed in relation to their interaction with branched-chain 2-oxo acid oxidation in incubated hemidiaphragms.     

Regulation of b- and a-Glycolytic Activities in the Sediments of a Eutrophic Lake

Temporal changes in α-and β-glucosidase activities, dissolved organic matter content, and bacterial biomass were studied in the superficial sediment layer of a eutrophic lake during the period of anoxia. The mean α-and β-glucosidase activities were 30.7±11.0 and 15.1±6.2 nmol h⁻¹ g⁻¹ of dry sediment, respectively. The specifc β-glucosidase activity seemed to be stimulated by carbohydrates (r=0.80, P<0.05), whereas the specifc α-glucosidase activity was negatively correlated with the dissolved protein concentration (r=−0.72, P<0.10). To test the effect of organic matter on hydrolytic activities under controlled conditions, changes in specific activities were studied in relation to the concentrations of different types of organic matter: phytoplankton, polymers (proteins, cellobiose, and starch) and monomers (glucose and amino acids). The specifc α-and β-glucosidase activities were strongly induced by their natural substrates (starch and cellobiose, respectively) (P<0.05) and were not inhibited by glucose. Proteins inhibited these activities (P<0.05), whereas supplementation with amino acids had no effect on specifc glycolytic activities.     

Microbial community development on model particles in the deep sulfidic waters of the Black Sea

Microorganisms attached to particles have been shown to be different from free-living microbes and to display diverse metabolic activities. However, little is known about the ecotypes associated with particles and their substrate preference in anoxic marine waters. Here, we investigate the microbial community colonizing particles in the anoxic and sulfide-rich waters of the Black Sea. We incubated beads coated with different substrates in situ at 1000 and 2000 m depth. After 6 h, the particle-attached microbes were dominated by Gamma- and Alpha-proteobacteria, and groups related to the phyla Latescibacteria, Bacteroidetes, Planctomycetes and Firmicutes, with substantial variation across the bead types, indicating that the attaching communities were selected by the substrate. Further laboratory incubations for 7 days suggested the presence of a community of highly specialized taxa. After incubation for 35 days, the microbial composition across all beads and depths was similar and primarily composed of putative sulfur cycling microbes. In addition to the major shared microbial groups, subdominant taxa on chitin and protein-coated beads were detected pointing to specialized microbial degraders. These results highlight the role of particles as sites for attachment and biofilm formation, while the composition of organic matter defined a secondary part of the microbial community.     

Some observations on the oxidation of glucose by enzymes in soil in the presence of toluene

Summary Changes in glucose concentrations were slight on incubation in a system of fresh soils, buffer, and toluene at 37°C and were not significantly detectable by a titrimetric method. Such changes would be too small to affect the accuracy of assays of enzymes hydrolysing carbohydrates in soil where activities are measured by glucose production.Oxygen uptake was slight but increased significantly when glucose was added to four undried soils incubated with toluene; uptake was greater at 37°C than at 24°C. Numbers of viable bacteria declined during incubation. Oxidation of glucose was negligible in a similar system with air-dried soils.Gluconic acid and 2-ketogluconic acid were identified as metabolic products from glucose incubated with soils and toluene. There was an approximate equivalence of oxygen uptake and acid production after incubation for 24 hours but not after longer periods. In the absence of toluene, both gluconic and 2-ketogluconic acids were readily metabolised by soils on incubation at 37°C.Results suggest that glucose oxidase and gluconate dehydrogenase are present in soils but that only a small proportion of glucose would be metabolised by oxidase activity in soils under natural conditions. Other oxidoreductase enzymes would also be active in soils.     

Survey of human virus occurrence in wastewater-recharged groundwater on Long Island.

Treated wastewater effluents and groundwater observation wells from three sewage recharge installations located on Long Island were assayed on a monthly basis for indigenous human enteroviruses and coliform bacteria for a period of 1 year. Viruses were detected in groundwater at sites where recharge basins were located less than 35 feet (ca. 10.6 m) above the aquifer. Results from one of the sites indicated the horizontal transfer of viable viruses through the groundwater aquifer.     

Survey of human virus occurrence in wastewater-recharged groundwater on Long Island.

Treated wastewater effluents and groundwater observation wells from three sewage recharge installations located on Long Island were assayed on a monthly basis for indigenous human enteroviruses and coliform bacteria for a period of 1 year. Viruses were detected in groundwater at sites where recharge basins were located less than 35 feet (ca. 10.6 m) above the aquifer. Results from one of the sites indicated the horizontal transfer of viable viruses through the groundwater aquifer.     

Enzyme Activities and Microflora of Earthworm Gut and Vermireactors as Indicators of the Stabilization of Waste Degradation Process

The worm and microorganisms act to enhance the biodegradation of organic matter. In earlier studies effect of enzymes has been studied in waste degradation process; however, no attempt has been made to determine the end point of organic waste degradation process through indication of hydrolases and dehydrogenase enzyme activity as well as microflora of earthworm and vermireactors. The present work is a pioneer endeavor in this direction. The study indicates that with time, hydrolase (β-glucosidase, N-x-benzoyl-l-argininamide [BAA]-hydrolyzing protease, urease, phosphatase, and cellulase) and dehydrogenase activities decrease as available organic compounds decreased. A high correlation among all enzyme activity and microflora was observed that led to the conclusion that activities of both enzymes (hydrolytic and dehydrogenase) and microflora could be the feasible indicators of organic matter degradation process.     

Quantitative Histochemistry; Design and Use of a Simple Microcell for Standardized Incubation on the Slide

Avoidance of stain variation among mounted sections incubated in small volumes of fluid can be overcome by the construction and use of a microcell. Perspex rings of appropriate diameter are attached to small Perspex rectangles to form wells, small trip bars being also attached. to the rectangles to simplify subsequent manipulation. The wells are filled with incubation fluid, and slides bearing frozen sections are held in position with small rubber bands. With certain dehydrogenases formazan production by this method is uniformly greater than that produced by incubation in drops of solution. In the case of the moderately active enzyme, 6-phosphogluconate dehydrogenase, the volume of the incubation mixture was not critical between 65 and 270 μl.