Microbiological response to well pumping
The data indicate that in response to increased well pumping, biologically sensitive parameters, and by inference, the subsurface microbiological community, evolved for at least 120 h. Prior to onset of the experiment, chemical and biological processes near the well may have been in a near steady-state condition, since the pump rate had been maintained in previous years at 0.5 L/min. Increasing the pumping rate disrupted conditions near the well, and, in response, chemical and biological processes there adjusted, leading to the observed change in the biologically sensitive parameters.
An alternative explanation is that these parameters varied as a result of macroscopic heterogeneity in the aquifer. If microbial communities and activities are distributed unevenly in the subsurface (Goldscheider et al. 2006), beginning to pump the well rapidly might have drawn in water from disparate portions of the aquifer, causing water composition to change with time. The aquifer system studied, however, is known to be quite homogeneous (Figure 1) (Kempton et al. 1982). More notably, such an interpretation does not offer a ready explanation of why the physical and chemical parameters stabilized relatively quickly whereas the biologically sensitive parameters changed more slowly.
Parameters commonly observed during well sampling—temperature, pH, D.O., ORP, and EC—stabilized within an hour of initiating pumping, consistent with previous studies. The concentrations of ferrous iron, sulfide, and sulfate took about 8 h to stabilize, in contrast, and a steady state was maintained for the duration of the experiment. Concentrations of H2 and CH4 also stabilized within 8 h but later began to drift again. These parameters are particularly sensitive to biological activity. Since the second adjustment period appears to result from changes to the microbial community composition near the well, it might be argued that the values observed after the first adjustment period, when the parameters stabilized at about 8 h, are the observations most representative of conditions in the bulk aquifer.
Results of this study suggest that common sampling practice, which calls for flushing three to five well volumes before samples are taken, may be sufficient for temperature, pH, D.O., ORP, and EC measurements. Considerably more time, however, is needed to obtain representative concentrations of ferrous iron, sulfide, sulfate, H2, and CH4. Considering that microbial metabolisms influence each of these analytes, the change of the concentrations over 8 h indicates that microbial activity near the wellbore is not the same as in the bulk aquifer. Measurements obtained after a short period of pumping, then, do not reflect the true nature of ground water in the aquifer as a whole.
In the case of H2, the concentration nearly doubled after 8 h, which corresponds to purging the wellbore about 36 times, compared to measurements made at less than 4 h of pumping. If normal sampling protocols that require purging the wellbore only five times were to be used, the mass of electron donor available in the ground water would be greatly underestimated.
Under anaerobic conditions, H2 is produced by fermentative microorganisms, which metabolize organic matter in the subsurface, and consumed by anaerobic respiration (Lovley and Goodwin 1988). In our experiment, as the pumping rate increased and the well was flushed with ground water, the H2 concentration increased rapidly, stabilizing after about 8 h (Figure 3A). This concentration may come close to reflecting the actual ambient conditions in the aquifer. By providing substrates like H2 to the vicinity of the wellbore, however, the pumping promotes microbial growth there, as reflected by the increase in microbial cell count between 50 and 120 h of pumping (Figure 3C). The range of microbial cell counts for the ground water is of the magnitude seen in other ground water studies (Stevens et al. 1993; Pedersen and Ekendahl 1990; Olson et al. 1981). Over the initial 55 h of pumping, the number of microorganisms ranged from 5 × 106 to 6 × 106 cells/L. Between 55 and 120 h, the total microbial numbers in the ground water samples increased to 9.4 × 106 cells/L (Figure 3C).
If we assume that methanogens are a dominant group of microorganism, we can estimate the group’s growth from the increase in CH4 concentration, which rose from 70 to 85 μM from 55 to 120 h. Based on known stoichiometric relationships associated with the coupling of methanogenesis to growth (Rittmann and McCarty 2001), given this increase, we would expect the population of methanogens to rise about 20%, a value significantly less than observed. The discrepancy may reflect the fact that planktonic microbial populations in ground water samples do not necessarily reflect microbial densities in subsurface systems (Alfreider et al. 1997) because most microorganisms in an aquifer live attached to solid surfaces. The result may also indicate that actively growing microorganisms are relatively easily detached from aquifer surfaces, contributing to their being overrepresented in the planktonic fraction.
After 30 h, the H2 concentration decreased and CH4 concentration increased (Figures 3A and 3B). The decrease of H2 could be explained in part by increased rates of hydrogentrophic methanogenesis:
This is the case even though the measured H2 concentration (35 to 100 nM) is in the range of the H2 threshold concentration for methanogenesis (5 to 95 nM) (Conrad 1996; Ralf et al. 1988; Lovley 1985; Lovley and Goodwin 1988). This reaction, however, can account for only a small fraction of the increase in CH4 concentration because more CH4 is gained than H2 lost (+15 μm vs. −60 nM). The bulk of the methane produced, therefore, may arise from acetoclastic methanogenesis:
Acetate can be produced by acetogenic bacteria in natural anaerobic environments as a byproduct of biomass decay (Conrad 1999), where it becomes available for use by acetoclastic methanogens. The dissolved organic carbon (DOC) concentrations in this ground water are 3.0 mg/L (Najm et al. 1993), which if present purely as acetate would yield 125 μM of CH4 by Reaction 2. The hydrogentrophic reduction of CO2 to acetate, another potential source of acetate, is not likely in this case since the H2 concentrations in this aquifer are too low for the reaction to occur (Dolfing 1988) and the amount of acetate possibly produced would account only for about 15 nM CH4 generation, 1/1000 of that observed.
The results of T-RFLP analysis in our study show that the bacterial community composition remained stable for the first 4 h (Figures 4 and 5). Between 30 and 50 h, the composition of the bacterial community was also stable; however, it differed considerably from the initial community. After 50 h, the community changed again as growth due to increased substrate flux became significant. Some members of the initial bacterial community reestablished themselves in the final stage of the experiment; however, the overall community profile differed considerably from the initial profile.
In contrast to the results for bacteria, the archaeal community of presumed methanogens was largely invariant throughout the experiment (Figure 6). This result is somewhat surprising because H2 consumption and CH4 production in the ground water (Figures 3A and 3B) indicate that methanogenesis increased. This could be interpreted in two ways: (1) acetoclastic methanogens are dominant and a small increase in the hydrogenotrophic populations may not significantly change the community structure or (2) all the methanogens in the community grew at approximately equal rates in response to the increased availability of H2 and potentially acetate. Since T-RFLP provides no indication of absolute biomass concentration, we were not able to tell which of the possibilities is more likely. This situation demonstrates the usefulness of using H2 and CH4 concentrations as an indicator of microbial activity. No clear change in archael T-RFLP patterns is apparent, but the change in H2 and CH4 concentrations seems to require an increased rate of methanogenesis.
In summary, variation in H2 and CH4 concentrations, T-RFLP profiles, and microbial cell counts demonstrate that there were two intervals of adjustment in the well over the course of the experiment. During the initial adjustment period, H2 and CH4 concentrations initially reflected conditions near the well. Concentrations immediately started to increase toward conditions in the bulk aquifer away from the well. Following a period of stability, there is a second period of acclimation marked by the increase in cell density and a change in the composition of the microbial community near the well.
Implications for sampling ground water
Chemical and biological analytes in the experiment evolved over four distinct time periods (Table 2). If we are concerned with only dissolved ions (e.g., ferrous iron, sulfide, and sulfate), the sampling can be done after displacement of about 36 well volumes (8 h of pumping this well). For dissolved ions, gas concentrations (e.g., H2 and CH4), and the microbial community, the optimum sampling interval continued until the wellbore had been displaced about 230 times (50 h for this well).
Stability of Physicochemical and Microbial Components of Ground Water Samples at Different Time Periods
|Water quality parameters|
| pH, ORP, alkalinity||—||—||—||—|
| D.O., temperature||↘||—||—||—|
|Microbial population (cells/L)||—||—||—||↗|
The optimum interval for obtaining a sample representative of these parameters can be expected to vary depending on well setting (e.g., the hydrogeologic environment and subsurface microbial community) and pumping rate. Our results, however, suggest that it can be identified by tracking when metabolic indicators such as H2, CH4, and Fe(II) stabilize, perhaps at around 36 wellbore displacements. These parameters may begin to change again in response to new microbial growth near the well, and the growth may be accompanied by a shift in the microbial community composition.