Groundwater Microorganisms Research Articles

Review of Factors Affecting Microbial Survival in Groundwater

This review quantitatively examines a number of published studies that evaluated survival and inactivation of public-health-related microorganisms in groundwater. Information from reviewed literature is used to express microbial inactivation in terms of log10 decline per day for comparison to other studies and organisms. The geometric mean value for inactivation rates for coliphage, poliovirus, echovirus, coliform bacteria, enterococci, and Salmonella spp. were similar at approximately 0.07-0.1 log10 day(-1), while geometric mean inactivation rates for hepatitis A virus, coxsackievirus, and phage PRD-1 were somewhat less at 0.02-0.04 log10 day(-1). Viruses show a temperature dependency with greater inactivation at greater temperatures; however this occurs largely at temperatures greater than 20 degrees C. Coliform bacteria die off in groundwater does not show the temperature dependency that viruses show, likely indicating a complex interplay of inactivation and reproduction subject to influences from native groundwater organisms, temperature, and water chemistry. The presence of native microorganisms seems to negatively impact E. coli survival more so than viruses, but in most cases, nonsterile conditions led to a greater inactivation for viruses also. The effect of attachment to solid surfaces appears to be virus-type-dependent, with PRD-1 more rapidly inactivated as a result of attachment and hepatitis A and poliovirus survival prolonged when attached.

Survival rate of pathogenic bacteria and viruses in groundwater

The results of an analytical review of publications on the process of inactivation of pathogenic microorganisms in groundwater are considered. The process of inactivation is described by a mathematical model based on an exponential dependence. The values of the inactivation rate factor for a number of pathogenic microorganisms are given. Recommendations are given regarding formulas for determining the dependence of the inactivation rate factor of bacteria and viruses on water temperature.

Bacterial community composition determined by culture‐independent and ‐dependent methods during propane‐stimulated bioremediation in trichloroethene‐contaminated groundwater

An in situ co-metabolic air sparging (CAS) study was carried out at McClellan Air Force Base (MAFB), Sacramento, CA, USA, in a trichloroethene- (TCE) and cis-dichloroethene (cis-DCE)-contaminated aquifer where one test zone received 2% propane in air and the other served as a control and received only air. As part of that study, bacterial population shifts were evaluated by length heterogeneity polymerase chain reaction (LH-PCR). The results showed that an organism(s) that had a fragment size of 385 bp was positively correlated with propane removal rates. The 385 bp fragment consisted of up to 83% of the total fragments in the analysis when propane removal rates peaked. A 16S rRNA clone library made from the bacteria sampled from the propane-sparged groundwater included clones of a TM7 division bacterium that had a 385 bp LH-PCR fragment; no other bacterial species with this fragment size were detected. Both propane removal rates and the 385 bp LH-PCR fragment decreased as nitrate levels in the groundwater decreased. Extinction culturing in natural unamended groundwater medium was used to assess the bacterial diversity of the culturable fraction of microorganisms in both CAS and air-sparged groundwater and to bring novel species into culture for further study. The dominant cultures acquired from the CAS groundwater were from the Herbaspirillum/Oxalobacter clade. The dominant cultures from the air-sparged groundwater were from a novel beta-Proteobacterial clade, which we named after isolate HTCC333.

The role of microbial community composition and groundwater chemistry in determining isoproturon degradation potential in UK aquifers

The community response of indigenous sandstone, chalk and limestone groundwater microorganisms to the addition of the commonly used herbicide isoproturon was examined. The addition of 100 μg l −1 isoproturon generally caused an increase in species diversity determined by chemotaxonomic analysis (fatty methyl ester analysis) of isolates resulting from incubation of cultures at 18 °C for 4 days. Amongst the groundwater samples to which isoproturon was added, isoproturon degradation rates were correlated with increasing dominance of a few species. However, the changes in community profile associated with isoproturon degradation varied from site to site. Repeated sub-culturing with 100 μg l −1 isoproturon and sterile groundwater was carried out to examine whether this level of pesticide could exert a selection pressure, and hence stimulate more rapid degradation. Significantly increased degradation was observed in a groundwater sample from the chalk, but not in sandstone, or limestone samples. The addition of filter-sterilised sandstone groundwater to bacteria on filter paper from slow degrading limestone sites significantly improved their degrading performance. The addition of filter-sterilised limestone groundwater to the sandstone bacteria reduced their degradation rate only slightly. The data suggested that the nature of the indigenous community does influence pesticide degradation in groundwater, but that the groundwater chemistry may also play a role.

Influence of groundwater characteristics on the survival of enteric viruses.

This study was undertaken to further understand the processes affecting the persistence of enteric viruses in groundwater. Varying temperature, oxygen and nutrient levels were tested in the presence and absence of groundwater micro-organisms to determine which of the factors tested had dominant influence on the decay of Escherichia coli, the bacteriophage MS2, poliovirus and coxsackievirus. The results indicated that the most influential factor affecting the decay of the viruses and E. coli was the presence of groundwater micro-organisms. The results also implied that temperature, the presence of oxygen and nutrient levels indirectly influence viruses and E. coli decay by influencing the activity of the groundwater micro-organisms. E. coli and the viruses displayed maximum decay under aerobic conditions, at 28 degrees C without the addition of nutrients in the presence of groundwater micro-organisms. The results suggest that if the mode of action of the groundwater micro-organisms could be determined then the decay of viral pathogens in recharged waters may be more easily predicted.

Metagenomic profiling: microarray analysis of an environmental genomic library.

Genomic libraries derived from environmental DNA (metagenomic libraries) are useful for characterizing uncultured microorganisms. However, conventional library-screening techniques permit characterization of relatively few environmental clones. Here we describe a novel approach for characterization of a metagenomic library by hybridizing the library with DNA from a set of groundwater isolates, reference strains, and communities. A cosmid library derived from a microcosm of groundwater microorganisms was used to construct a microarray (COSMO) containing approximately 1-kb PCR products amplified from the inserts of 672 cosmids plus a set of 16S ribosomal DNA controls. COSMO was hybridized with Cy5-labeled genomic DNA from each bacterial strain, and the results were compared with the results for a common Cy3-labeled reference DNA sample consisting of a composite of genomic DNA from multiple species. The accuracy of the results was confirmed by the preferential hybridization of each strain to its corresponding rDNA probe. Cosmid clones were identified that hybridized specifically to each of 10 microcosm isolates, and other clones produced positive results with multiple related species, which is indicative of conserved genes. Many clones did not hybridize to any microcosm isolate; however, some of these clones hybridized to community genomic DNA, suggesting that they were derived from microbes that we failed to isolate in pure culture. Based on identification of genes by end sequencing of 17 such clones, DNA could be assigned to functions that have potential ecological importance, including hydrogen oxidation, nitrate reduction, and transposition. Metagenomic profiling offers an effective approach for rapidly characterizing many clones and identifying the clones corresponding to unidentified species of microorganisms.

東濃地域における地下水化学と地下微生物の相互作用

The abundance and diversity of groundwater microorganisms were studied in a geochemically defined borehole (TH-6, DH-3) in the Tono area, central Japan. Total cell counts by epifluorescence microscopy were estimated to be approximately 1.6 × 105-5.7 × 106 cells ml -1 and showed little decrease with depth. Cell viability, based on cell membrane integrity, respiration-based metabolism, and esterase activity was estimated to be 0.001% to approximately 100% of the total counts. The distribution of microbial abundance seems to be related to a variety of environmental factors, including fracture numbers, hydrological, and geochemical conditions of the groundwater. The geochemistry of the groundwater suggests that sulfate reducing bacteria play an important role in the sedimentary rock environment. It appears that microbial sulfate reduction occurs at the highest level in the upper part of the Toki Lignite-bearing Formation. This microbial reaction may play an important role in maintaining anaerobic conditions for uranium fixation, that could in turn scavenge soluble and oxidized uranium. On the other hand, in the granite groundwater, iron-oxidizing/reducing bacteria seem to play an important role in iron redox cycling. Iron-oxidizing bacteria may contribute to the formation and deposition of iron colloids in the upper part of Toki granite.

Persistence of biofilm-associated Escherichia coli and Pseudomonas aeruginosa in groundwater and treated effluent in a laboratory model system.

This study was based on the hypothesis that groundwater-derived biofilms may provide a reservoir for coliform or pathogenic bacteria as has been observed in drinking water distribution systems. Escherichia coli, labelled with green fluorescent protein, was found to colonize all layers of mixed-population biofilms developed in association with indigenous groundwater micro-organisms in a laboratory-scale reactor. Biofilm-associated E. coli was removed at a slower rate from the reactor flasks than planktonic E. coli under a continuous flow regime. During flow-through of groundwater, planktonic E. coli removal was slower in flasks containing coverslips for enhanced biofilm development compared to a control flask without coverslips. Conversely, during flow-through of treated effluent, planktonic E. coli removal was faster in flasks with coverslips compared to without. Removal of attached E. coli was also fastest in the coverslip-containing flasks with effluent flow-through. This suggests that an increase in available nutrients may reduce E. coli survival potential due to either enhanced competition for nutrients or enhanced antagonism by the indigenous microbial population. Under identical conditions, GFP-labelled Pseudomonas aeruginosa was found to persist in the biofilms for longer than E. coli, most notably when exposed to flow-through of treated effluent. However, prolonged persistence of P. aeruginosa in the effluent could not be attributed to an association with the biofilms. This study has shown that under certain conditions the presence of mixed-population biofilms may limit the survival potential of enteric bacterial pathogens introduced into groundwater.

Abundance and Viability of the Groundwater Microbial Communities from a Borehole in the Tono Uranium Deposit Area, Central Japan.

The abundance and viability of microorganisms in groundwater were studied using a scientific borehole in the Tono uranium deposit area, central Japan. Groundwater samples were collected from scientific borehole TH-6 at four depths; 104, 132 and 153 m in sedimentary rocks and 177 m in granite rock using autoclaved geochemical water samplers. The total cell count using epifluorescence microscopy was of the order of 105 to 106 cells ml-1, showing non-systematic change in microbial parameters with depth. Viability estimated from cell membrane integrity ranged from 22.3% to almost 100%, and showed a tendency to increase with depth. In contrast, viability determined using both activities of the electron transport system (ETS) and esterase showed inconsistent depth-profiles. The ETS-active cell count corresponded to 0.57-24.7% of the total count; the minimum ETS-count was found at 153 m, just above the sediment-granite unconformity where the minimum redox potential was estimated. The esterase-active cell count corresponded to 0.4-10.3% of the total, with a maximum at 132 m, the lignite-derived organic-rich layer, and a minimum was found at 153 m, as seen for the ETS-active counts. These inconsistent profiles suggest a difference in microbial viability and activity between each depth. In addition, depth-specific microflorae may develop in response to the availability of various electron acceptors and donors in the subsurface.

Assessment of Changes in Biodiversity when a Community of Ultramicrobacteria Isolated from Groundwater Is Stimulated to Form a Biofilm.

The stimulation of groundwater bacteria to form biofilms, for the remediation of polluted aquifers, is subjected to environmental regulations that include measurement of effects on microbial biodiversity. Groundwater microorganisms contain a proportion of unidentified and uncharacterized ultramicrobacteria (UMB) that might play a major role in the bioclogging of geological materials. This study aimed to assess the changes in genetic and metabolic biodiversity when a community of UMB, isolated from groundwater, is stimulated to form biofilms on a ceramic surface. UMB were stimulated with aerobic conditions and injection of molasses, in reactors reproducing groundwater composition and temperature. Concentration of planktonic viable UMB, secretion of extracellular polymeric substances (EPS), and biofilm thickness were monitored. The assessment of changes in biodiversity was achieved by comparing the initial UMB community to the biofilm community, using the single strand conformational polymorphism (SSCP) method, the cloning and sequencing of 16S rRNA gene (16S rDNA) sequences, and the Biolog microplate system. The hypothesis stating that indigenous UMB would play a significant role of in the biofilm development was corroborated. Within 13 days of stimulation, the UMB produced 700 mg L?1 of planktonic EPS and formed a biofilm up to a thickness of 1100 mm. This stimulation led to a decrease in genetic diversity and an increase in metabolic diversity. The decrease in genetic diversity was shown by a reduced number of single strand DNA fragments in the SSCP profiles. As such, 16S rDNA sequences from the biofilm revealed the predominance of four bacterial groups: Zoogloea, Bacillus/Paenibacillus, Enterobacteriaceae, and Pseudomonads. A significant increase in metabolic diversity was shown by a highest substrate richness profile and a lower substrate evenness profile of the biofilm bacterial population (p = 0.0 and p = 0.09, respectively). This higher metabolic diversity might be a consequence of the stimulation that seemed to favor the growth of bacteria having a high nutritional versatility. Stimulation of UMB, isolated from groundwater, was effective to form a biofilm having a high metabolic biodiversity. This combination of molecular-based and metabolic-based methods expanded the insight into monitoring the changes in bacterial biodiversity.

Naphthalene and donor cell density influence field conjugation of naphthalene catabolism plasmids.

We examined transfer of naphthalene-catabolic genes from donor microorganisms native to a contaminated site to site-derived, rifampin-resistant recipient bacteria unable to grow on naphthalene. Horizontal gene transfer (HGT) was demonstrated in filter matings using groundwater microorganisms as donors. Two distinct but similar plasmid types, closely related to pDTG1, were retrieved. In laboratory-incubated sediment matings, the addition of naphthalene stimulated HGT. However, recipient bacteria deployed in recoverable vessels in the field site (in situ) did not retrieve plasmids from native donors. Only when plasmid-containing donor cells and naphthalene were added to the in situ mating experiments did HGT occur.

The transport and behaviour of isoproturon in unsaturated chalk cores

A batch sorption study, a microcosm degradation study, and two separate column leaching studies were used to investigate the transport and fate of isoproturon in unsaturated chalk. The column leaching studies used undisturbed core material obtained from the field by dry percussion drilling. Each column leaching study used 25 cm long, 10 cm wide unsaturated chalk cores through which a pulse of isoproturon and bromide was eluted. The cores were set-up to simulate conditions in the unsaturated zone of the UK Chalk aquifer by applying a suction of 1 kPa (0.1 m H 2O) to the base of each column, and eluting at a rate corresponding to an average recharge rate through the unsaturated Chalk. A dye tracer indicated that the flow was through the matrix under these conditions. The results from the first column study showed high recovery rates for both isoproturon (73–92%) and bromide (93–96%), and that isoproturon was retarded by a factor of about 1.23 relative to bromide. In the second column study, two of the four columns were eluted with non-sterile groundwater in place of the sterile groundwater used on all other columns, and this study showed high recovery rates for bromide (85–92%) and lower recovery rates for isoproturon (66–79% — sterile groundwater, 48–61% — non-sterile groundwater). The enhanced degradation in the columns eluted with non-sterile groundwater indicated that groundwater microorganisms had increased the degradation rate within these columns. Overall, the reduced isoproturon recovery in the second column study was attributed to increased microbial degradation as a result of the longer study duration (162 vs. 105 days). The breakthrough curves (BTCs) for bromide had a characteristic convection–dispersion shape and were accurately simulated with the minimum of calibration using a simple convection–dispersion model (LEACHP). However, the isoproturon BTCs had an unusual shape and could not be accurately simulated.

Aerobic biodegradation of dichloroethylenes in surface and subsurface soils

Laboratory studies were conducted to examine the aerobic biodegradation of dichloroethylenes (cis-1,2-DCE, trans- 1,2-DCE and 1,1-DCE) in soil and groundwater. Authentic surface and subsurface materials with no reported DCE exposure history were used. All DCE isomers were observed to biodegrade to varying degrees in the soils examined. Use of radiolabeled [14C] test chemicals allowed correlation of DCE disappearance with mineralization to 14CO2. Study. results indicate that naturally occurring microorganisms in soil and groundwater are capable of degrading cis-1,2-, trans-1,2- and 1,1-DCE without laboratory supplementation of exogenous organic nutrients, or previous exposure history. The data further suggest that degradative potential may vary with soil type, DCE isomer structure, and concentration.

In situ, real-time catabolic gene expression: extraction and characterization of naphthalene dioxygenase mRNA transcripts from groundwater.

We developed procedures for isolating and characterizing in situ-transcribed mRNA from groundwater microorganisms catabolizing naphthalene at a coal tar waste-contaminated site. Groundwater was pumped through 0.22-microm-pore-size filters, which were then frozen in dry ice-ethanol. RNA was extracted from the frozen filters by boiling sodium dodecyl sulfate lysis and acidic phenol-chloroform extraction. Transcript characterization was performed with a series of PCR primers designed to amplify nahAc homologs. Several primer pairs were found to amplify nahAc homologs representing the entire diversity of the naphthalene-degrading genes. The environmental RNA extract was reverse transcribed, and the resultant mixture of cDNAs was amplified by PCR. A digoxigenin-labeled probe mixture was produced by PCR amplification of groundwater cDNA. This probe mixture hybridized under stringent conditions with the corresponding PCR products from naphthalene-degrading bacteria carrying a variety of nahAc homologs, indicating that diverse dioxygenase transcripts had been retrieved from groundwater. Diluted and undiluted cDNA preparations were independently amplified, and 28 of the resulting PCR products were cloned and sequenced. Sequence comparisons revealed two major groups related to the dioxygenase genes ndoB and dntAc, previously cloned from Pseudomonas putida NCIB 9816-4 and Burkholderia sp. strain DNT, respectively. A distinctive subgroup of sequences was found only in experiments performed with the undiluted cDNA preparation. To our knowledge, these results are the first to directly document in situ transcription of genes encoding naphthalene catabolism at a contaminated site by indigenous microorganisms. The retrieved sequences represent greater diversity than has been detected at the study site by culture-based approaches.

Microorganisms as tracers in groundwater injection and recovery experiments: a review.

Modern day injection and recovery techniques designed to examine the transport behavior of microorganisms in groundwater have evolved from experiments conducted in the late 1800s, in which bacteria that form red or yellow pigments were used to trace flow paths through karst and fractured-rock aquifers. A number of subsequent groundwater hydrology studies employed bacteriophage that can be injected into aquifers at very high concentrations (e.g., 10(13) phage ml-1) and monitored through many log units of dilution to follow groundwater flow paths for great distances, particularly in karst terrain. Starting in the 1930s, microbial indicators of fecal contamination (particularly coliform bacteria and their coliphages) were employed as tracers to determine potential migration of pathogens in groundwater. Several injection and recovery experiments performed in the 1990s employed indigenous groundwater microorganisms (both cultured and uncultured) that are better able to survive under in situ conditions. Better methods for labeling native bacteria (e.g. by stable isotope labeling or inserting genetic markers, such as the ability to cause ice nucleation) are being developed that will not compromise the organisms' viability during the experimental time course.

Biodegradation of NSO-compounds under different redox-conditions

Laboratory experiments were carried out to investigate the potential of groundwater microorganisms to degrade selected heterocyclic aromatic compounds containing nitrogen, sulphur, or oxygen (NSO-compounds) under four redox-conditions over a period of 846 days. Eight compounds (pyrrole, 1-methylpyrrole, quinoline, indole, carbazole, dibenzothiophene, benzofuran, and dibenzofuran) were degraded under aerobic conditions, whereas thiophene and benzothiophene were degraded only when other compounds were degraded concomitantly. Quinoline and indole were the only two NSO-compounds degraded under anaerobic conditions, even though the microorganisms present in the anaerobic microcosms were active throughout the incubation period. A high variability in the lag period among the NSO-compounds was observed under aerobic conditions. While quinoline, indole, and carbazole were degraded with a lag period of 3–25 days, the lag periods for pyrrole, dibenzothiophene, benzofuran, and dibenzofuran were significantly longer (29–278 days). Under anaerobic conditions, lag periods of 100–300 days were observed. Differences in the degradation rate among the compounds were also observed. Indole, quinoline, carbazole, and benzofuran were quickly degraded in the aerobic microcosms, whereas a slow degradation of dibenzothiophene and dibenzofuran was observed. Pyrrole and 1-methylpyrrole were slowly degraded and 1-methylpyrrole was not completely removed within the 846 days. The anaerobic degradation rate was significantly slower than the aerobic degradation rate. The degradation rate under sulphate-reducing conditions was higher than under denitrifying and methanogenic conditions, though after re-addition of a compound a quick removal was observed. The persistence of many NSO-compounds under anaerobic conditions together with the long lag periods and the low degradation rates under aerobic conditions suggest that NSO-compounds might persist in groundwater at creosote-contaminated sites.

Biodegradation of thiophene, benzothiophene, and benzofuran with eight different primary substrates

AbstractThe aerobic biodegradation of thiophene, benzothiophene, and benzofuran was studied in microcosm experiments using groundwater microorganisms as inoculum. Benzofuran, a heterocyclic aromatic compound containing oxygen, was biodegraded as a sole source of carbon and energy measured by the disappearance of the compound, whereas two heterocyclic aromatic compounds containing sulphur (thiophene and benzothiophene) were not used as growth substrates. Thiophene was biodegraded with benzene, toluene, and to some extent ethylbenzene as primary substrates. Some biodegradation of thiophene was observed when p‐xylene, o‐xylene, m‐xylene, naphthalene, and 1‐methylnaphthalene were the primary substrates. Benzothiophene was completely transformed, with all eight primary substrates investigated except for benzene and p‐xylene, in which 34 and 6% of benzothiophene, respectively, remained after 40 d of incubation. Although benzofuran could be used as a sole source of carbon and energy, data showed that the biodegradation of benzofuran definitely was enhanced by the biodegradation of the primary substrates used.

In-situ bioremediation of an aquifer contaminated with 1,2-dichloroethane

This article describes the application of in-situ bioremediation for the treatment of an aquifer contaminated with 1,2-dichloroethane (DCA). The first step in the process was to properly delineate the contamination and to contain the contaminated groundwater using a pumping well. The second step was to isolate in the groundwater microorganisms able to degrade DCA and to demonstrate the possibility of increasing their efficiency by injecting in-situ nutrients and hydrogen peroxide (H2O2) solution. In the third step, after the characterization of the hydrogeology of the aquifer with tracing experiments, the in-situ bioremediation of the groundwater was conducted. The analyses show that 95 percent of DCA was destroyed by this treatment, leading to a DCA concentration around the pumping well of about 0.2 mg/l.

Microcosm and in situ field studies of enhanced biotransformation of trichloroethylene by phenol-utilizing microorganisms

The ability of different aerobic groundwater microorganisms to cometabolically degrade trichloroethylene (TCE), 1,2-cis-dichloroethylene (c-DCE), and 1,2-trans-dichloroethylene (t-DCE) was evaluated both in groundwater-fed microcosms and in situ in a shallow aquifer. Microcosms amended with phenol or toulene were equally effective in removing c-DCE (> 90%) followed by TCE (60 to 70%), while the microcosm fed methane was most effective in removing t-DCE (> 90%). The microcosm fed ammonia was the least effective. None of the microcosms effectively degraded 1,1,1-trichloroethane. At the Moffett Field groundwater test site, in situ removal of c-DCE and TCE coincided with biostimulation through phenol and oxygen injection and utilization, with c-DCE removed more rapidly than TCE. Greater TCE and c-DCE removal was observed when the phenol concentration was increased. Over 90% removal of c-DCE and TCE was observed in the 2-m biostimulated zone. This compares with 40 to 50% removal of c-DCE and 15 to 25% removal of TCE achieved by methane-grown microorganisms previously evaluated in an adjacent in situ test zone. The in situ removal with phenol-grown microorganisms agrees qualitatively with the microcosm studies, with the rates and extents of removal ranked as follows: c-DCE > TCE > t-DCE. These studies demonstrate the potential for in situ TCE bioremediation using microorganisms grown on phenol.

Bioremediation of groundwater pollution.

Significant progress has been made in the past year towards an understanding of the microbial processes in subsurface environments that may allow natural microbial populations to be employed for bioremediation of groundwater pollution. Among the highlights were: the discovery of several previously unknown xenobiotic-degrading abilities in groundwater microorganisms; progress in using the unique abilities of methanotrophs to oxidize halogenated solvents; and characterizations of microbial populations from subsurface soils.