World Journal of Microbiology & Biotechnology 10, 325-333
Decrease of the hydraulic conductivity of sand columns by Methanosarcina barkeri D. Sanchez de Lozada, P. Vandevivere, P. Baveye* and S. Zinder The extent to which a methanogen can clog sand columns was examined: two permeameters packed with clean quartz sand were sterilized, saturated with water, inoculated with Methanosarcina barkeri and percolated under upward flow conditions. After approx. 5 months, the hydraulic conductivity of the sand had decreased to 3% and 25% of the highest values measured earlier. At that point, gas-filled regions in the sand were clearly visible through the transparent walls of the permeameters, and methane bubbles were continuously released from the columns into the effluent. Scanning electron microscopy observations and biomass assays indicated that cell mass accumulation did not contribute significantly to the observed decrease of the hydraulic conductivity. This decrease was therefore attributed to pore blocking due to the entrapment of methane bubbles. Key words: Aquifer materials, clogging, hydraulic conductivity, methane production, methanogenesis, pore blockage.
In the last few decades, the clogging of waterlogged soils and aquifer materials has been the object of a sustained research effort, primarily because of the adverse effects it has on a number of processes, such as the disposal of waste water (Davis et al. 1973; Chang et al. 1974), groundwater recharge (Ripley & Saleem 1973; Oberdorfer & Peterson 1985), groundwater production (Van Beek 1984) or the in situ bioremediation of contaminated aquifer sediments (Lee et al. 1988). The clogging of saturated soils and aquifer materials is manifested by an often drastic decrease in their ability to transmit water, i.e. of their saturated hydraulic conductivity, H C s a t . The various mechanisms suggested to account for this phenomenon are usually classified as physical, chemical or biological. Physical mechanisms include the filtration of solid particles suspended in the percolating liquid, and the progressive disintegration of the soil structure. Chemical phenomena that affect the H C t involve changes in the swelling properties of soils and the dispersion of colloidal particles brought about by variations in the composition of the liquid phase. The mechanisms by which living organisms influence the HCs, t of
O. Sanchez de Lozada and P. Baveye are with the Department of Soil, Crop
and Atmospheric Sciences, Bradfield Hall, Comen University, Ithaca, NY 13853, USA; fax: 607 255 2644. P. Vandevivere is with the College of Marine Studies, University of Delaware, Lewes, DE 19958, USA. S. Zinder is with the Department of Microbiology, Rice Hall, CorneU University, Ithaca, NY 14853, USA. * Corresponding author.
soils and aquifers are by far the least understood. The microflora that are involved in this process consist predominantly of bacteria (Van Beek 1984; Shaw et al. 1985) although fungi (Ripley & Saleem 1973; Okubo & Matsumoto 1983) and protozoa (Hilton & Whitehall 1979) may sometimes be involved. The mechanism most often advanced to account for the observed bacterial reduction of H C t in soils and aquifer materials involves the production of extracellular polymers (Allison 1947; Shaw et al. 1985). Vandevivere & Baveye (1992a) showed, however, that the production of extracellular polymers was not necessary for severe bacterial clogging to occur. Other mechanisms that may come into play and have been invoked occasionally in the literature include plugging of the pores by the bacterial cells themselves (Gupta & Swartzendruber 1962), precipitation in soil pores of iron and magnesium sulphides resulting from the activity of sulphate-reducing bacteria (Ford & Beville 1968; Van Beek 1984), deposition of iron hydroxides or manganese oxides produced by iron bacteria (Van Beek 1984), or the production of gas (Ahmad 1963), in particular by denitrifiers (Lance & Whisler 1972; Oberdorfer & Peterson 1985) and possibly by methanogens (Swartzendruber & Gupta 1964; Gupta & Swartzendruber 1964; Reynolds et al. 1992). To control the extent and intensity of bacterial clogging in sub-surface environments, more detailed information is
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required on the various mechanisms listed above than is currently available. In particular, a fundamental understanding is needed of the mechanisms that could cause the clogging of porous media by methanogens, which are ubiquitous in sub-surface environments (Lovley & Phillips 1987; Ghiorse & Wilson 1988; Stevens et al. 1993). One of the mechanisms by which methanogens could significantly affect HCsa t is via the release of methane. Takai et al. (1956) measured rates of methane production as high as 8 m l / d a y . g soil in a submerged paddy soil under static conditions. On the basis of a methane concentration of 3% ( v / v ) at saturation (Swartzendruber & Gupta 1964), such a production would lead to supersaturation in methane and possible bubble formation when the rate of water transport in the soil was less than approximately 6 x 1 0 -s m / s , a situation that is of widespread occurrence. This observation may explain the fact that the gas phase in ponded softs often contains significant amounts of methane (Kristiansen 1981; Tollner et al. 1983). Once the soil solution has become supersaturated, entrapment of methane bubbles may lead to pore blocking and, consequently, to a reduction of the hydraulic conductivity of the medium. The experimental evidence that is available on this process is still largely inconclusive. On the basis of the results of percolation experiments in sand columns, Swartzendruber & Gupta (1964) concluded that the production of methane by microorganisms could not cause clogging. This statement appears, however, unwarranted in view of the fact that no methanogens were involved in the experiments and that the solutions percolated through the sand were undersaturated with respect to methane, making the formation of bubbles in the pore space very unlikely. More recently, Reynolds et al. (1992) monitored the m o v e m e n t of de-aired, temperature-equilibrated water through repacked laboratory columns of catotelm peat. In unsterilized columns, the hydraulic conductivity decreased sharply over a period of 78 days, while, concomittantly, the volumetric gas content and the gaseous methane concentration in the peat increased significantly. These observations suggest that in some cases there may be a strong correlation between in situ accumulation of anaerobe-generated methane and severe decreases in hydraulic conductivity. However, since Reynolds et al. (1992) did not investigate other possible mechanisms (e.g. biomass accumulation, extracellular polymer production) by which methanogens could clog the porous media, one cannot deduce from the existence of this correlation that there is a causal link between methane production and decreases in hydraulic conductivity. In the present study, laboratory experiments were conducted to appraise the extent to which a methanogen, Methanosarcina barkeri, could decrease the hydraulic conductivity of sand columns. A second objective of these experiments was to elucidate the mechanisms responsible for the observed clogging.
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Materials and Methods Bacterial Strain and Nutrient Solution The bacterial strain used in the experiments was originally isolated from sewage sludge (Mah et al. 1978) and is classified as Methanosarcina barkeri 227, a strict anaerobe that can use a number of substrates: H2/CO2, methanol acetate, formate or methylamines (Blaut et al. 1985; Jones et al. 1987). The deoxygenated solution used to grow the bacterium in batch cultures, which was also percolated through the sand columns, contained (rag/1 deionized water): methanol, 1483; NH4C1, 500; KH2PO4, 740, K2HPO4, 200; MgCI2. 6H20, 100; NAG1, 667; NaHCO 3, 37.5; Na2S.9H20, 281; and CaC12, 45; in addition to 0.875 ml of Resazurin 0.1%/1 and 10 ml of a trace element stock solution/1 (Zeikus 1977; Lobo & Zinder 1988). The phosphate buffer maintained the pH of the solution at 6.8. The preparation method was identical to that adopted by Lobo & Zinder (1988) except for a change in buffer and the fact that the nutrient solution was kept under a pure N 2 atmosphere rather than under a 70% N2/30% CO2 (v/v). Further details on the preparation of the nutrient solution are given by Sanchez de Lozada (1992). Sand The sand used in this study originated from the same site in Arenzville (Cass County, IL, USA) as that used by Vandevivere & Baveye (1992a,b,c). It contains quartz (92%) and magnetite (8%). The particle size range was narrowed down to between 63 and 125 mm by sieving. The sand was washed with sodium acetate (pH 5) to remove carbonates, H202 (6%) to remove organic matter and a mixture of sodium citrate, NaHCO 3 and sodium hydrosulphite to remove iron oxides, autoclaved for 90 min and dried at 105°C. Flow System The flow system is represented schematically in Figure 1. The nutrient solution, held aseptically in 10-1 Pyrex bottles that functioned as Mariotte devices, was percolated under constant hydraulic head conditions in permeameters packed with sand. Two such permeameters were operated simultaneously (columns L and R). Each consisted of an acrylic cylinder (70 mm in length and 25 mm inside diameter) capped at both ends with headpieces manufactured with a conical chamber to ensure an axial flow within the sand and to facilitate the entrainment of gas bubbles. Rubber O-rings provided a good seal at the contact between the acrylic cylinder and the headpieces. The sand was confined within the cylinder by nylon meshes (53-~tm pore size), which in turn were supported by coarser steel meshes. Packing of the sand in the permeameters was achieved by gently tapping the plexiglass cylinder a few times after each addition of a 5 mm layer. Once packed in the column, the sand had a porosity (volume of voids divided by total volume) of 0.39 _+0.01. Except for the columns, which were sterilized by overnight exposure to ethylene oxide, all parts in contact with the flowing solution were autoclaved and assembled aseptically in a laminar-flow hood. The sand was slowly saturated from beneath and a steady upward flow of, autoclaved water was maintained for 7 days, until entrapped air was removed. Each column was then inoculated by injecting, through the septa of the inoculation ports (Figure 1), 10 ml of an exponentially-growing culture of M. barkeri. During the inoculation and shortly thereafter, the flow of solution
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Figure 1. Schematic diagram of the flow system. to the columns was stopped to foster cell attachment. The flow experiments were conducted at room temperature (20 +_3°C). Since M. barkeri 227 is extremely sensitive to the presence of 02, it was necessary to conduct the percolation experiments under anoxic conditions. To this end, a N2-cylinder and a set of tubes and connectors were used to maintain a N 2 atmosphere in the head space in the nutrient solution bottles and above the water columns in the piezometers (Figure 1). The tubing connecting the solution bottles to the permeameters was made of butyl rubber in order to decrease the diffusion of 02 in the flow system. Samples of the permeameter effluents were taken at 7-day intervals through the sampling ports (Figure 1) to measure changes in the concentration of dissolved methane by GC. The presence of a gas-trap (Figure 1), a piece of glass tubing of known internal diameter terminated by a rubber septum, made it feasible to monitor the volume and composition of the gas bubbles escaping at the top of the sand column. The volume of the gas in the trap was calculated on the basis of the position of the water
meniscus in the glass tube. Once the trap was filled, the gas was evacuated through the spetum with a sterile syringe. Upon initiation of bubble formation, as well as toward the end of the experiments, the relative methane content in the gas was measured by GC. Measurement The hydraulic conductivity (HCsa,) can be calculated from measurements of the hydraulic head gradient using Darcy's equation (Jury et aL 1991) HCsa t
AH
Q=AxHC
tx Az
where Q is the flow rate, A is the cross-sectional area of the sand column, H is the hydraulic head (water potential per unit weight), and Az is the distance between the two points where H
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D. Sanchez de Lozada et al. is measured. The head difference between the two points, AH, is the difference in elevation of water columns held in small tubings, called piezometers, connected to these points at one end and open to the atmosphere (in this case, to a N 2 atmosphere) at the other end. These piezometers, in duplicates at each depth, protruded horizontally about 2 mrn inside the sand columns and were capped with nylon mesh (secured with tygon tubing) to prevent the sand from creeping in. They were inserted at five locations in the permeameters (see Figure 1); just below the inlet mesh, at 10, 20, and 50 nun above the inlet mesh and, finally, above the outlet mesh. Thus HC~t could be calculated for four different layers in the sand columns. In order to facilitate the analysis of the results, H C t values for each column were divided by the initial value (HCsat(t_o),) SO that the conductivity ratio (HCt/HCt(t=o)) was 1 at the time of inoculation. The average value ofHC sat(t-o) was 5.55 x 10-3(± 0.65 x 10-3) cm/s, corresponding to a flux density of approximately 8.2 cm/s. In the remainder of this article, we shall use the notation HCsat for the hydraulic conductivity even after providing evidence that a portion of the sand columns were not completely water-filled during the experiments. The subscript 'sat' should then be interpreted as indicating that the water was under a positive hydraulic head throughout the columns, i.e. was not retained in the sand by capillary action.
Gas Chromatography The analysis of methane in the permeameter effluents and gastraps was performed with a 5890A gas chromatograph (Hewlett Packard), using a 3.1-m Hayesep Q packed column connected to a H 2 flame ionization detector. Nitrogen was used as the carrier gas, flowing at 30 ml/min. The temperature was set at 130°C for the injector, oven and detector. Samples of the gas accumulated in the gas-traps were taken using 1 ml B-D Glaspak tuberculin syringes fitted with Mininert syringe valves, as described in Lobo & Zinder (1988). The same equipment was used for samples from the natural gas supply, used as standards. For the samples of effluent taken from the sampling port, the syringe valve was replaced with sterile 0.2-~tm pore Gleman Acrodisc syringe filters to avoid injection of bacteria into the GC columns.
Biomass Assay and Scanning Electron Microscopy After the flow was stopped at the end of the experiment, each permeameter was dismantled. The sand contained in the plexiglass cylinders was removed in successive layers of 10 mm thickness, after taking samples for scanning electron microscopy (SEM) observations. Using the method developed by Vandevivere & Baveye (1992d), two samples were taken at the inflow side of each of the sections corresponding to the distance intervals 0 to 10, 20 to 30, 40 to 50 and 60 to 70 mm from the column inlet, and were prepared for SEM using a glutaraldehyde/lysine mixture for fixation of the bacterial cells (Vandevivere & Baveye 1992e). Observations were made at three depths in each sample, at approximately 2 mm intervals, starting at the upstream side. The remaining sand from each layer was used to determine the average biomass density. The lipid-bound phosphate (in nmol) of the bacteria in the sand was determined by the assay of Findlay et al. (1989). The number of bacteria per volume of sand (X) was estimated from the resulting data by comparison with the lipid-bound phosphate extracted from a volume of a suspension with a known cell concentration. The mass per cell (Y) was obtained by estimating the average cell volume (on a wet mount under a light microscope), and by multiplying this volume by a
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wet density of 1.1 m g / m m 3, as described in Vandevivere & Baveye (1992a). An estimate of the biomass density was then obtained by taking the product of X and Y.
Results and D i s c u s s i o n The first manifestation of the activity of Methanosarcina barkeri 227 in the columns was a progressive increase in the concentration of methane dissolved in the column effluents (Figure 2). After approximately 100 days of flow for both columns, the methane concentration was within experimental error of the saturation level. Under equilibrium conditions, this would normally have marked the onsert of bubble formation. However, visual evidence suggested that, perhaps as a result of internal mixing, the increase in methane concentration in the effluents lagged behind that occurring in localized regions in the columns. Indeed, on day 89, bubbles started forming in one of the two piezometers situated 10 m m above the inlet mesh in column L. Simultaneously, and at approximately the same level in the column, entrapped gas was clearly visible through the transparent plexiglass walls of the permeameter (see Figure 3). Initially, the regions occupied by entrapped gas were small. However, they tended to coalesce progressively, increasing in size and eventually invading the piezometers which rapidly became inoperative in spite of frequent flushing and had to be closed off (on day 115). Desaturation in column R followed a similar pattern except that it started later (on day 97) and, throughout the experiment, took place predominantly within 20 m m of the outlet end of the sand column. Bubbles also formed in the inlet and outlet chambers of both permeameters. Their appearance coincided with the onset of gas accumulation in the piezometers. The bubbles forming in the inlet chamber rapidly coalesced into one large enough to create a gas interface between the bottom of the sand column and the incoming liquid, eventually preventing flow. Therefore it became necessary to evacuate the bubbles periodically from the inlet chambers of both permeameters. The bubbles in the outlet chambers seemed to form within the wire mesh. They grew progressively until they detached from the mesh and moved toward the brass connectors at the outlet end of the permeameters (see the bubble at the top of Figure 3) where they attached to the plexiglass walls until their diameter increased to such an extent that buoyancy forced them out of the permeameters and into the gas-traps. Soon after the beginning of the percolation experiments, the overall (column-averaged) hydraulic conductivity of the columns began to decrease (Figure 4). After a few weeks, it stabilized at approximately 85% of its original value. Given that methanogenic activity was extremely low during the first 40 days of flow, this initial decrease could not have been used by the bacterial population in the columns and may have been due to settling or rearrangement of the sand particles. However, no direct evidence is available to prove
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HCa , had dropped to 3% of its original value. In column R, which was dismantled slightly later (on day 151 ), the H C t was much higher, with a ratio of 0.25. In column L, piezometer readings (data not shown) revealed that the reduction in hydraulic conductivity was most pronounced in the inlet layer (0 to 1 cm). The HCs, t of the second layer (1 to 3) cm decreased rapidly between days 85 and 95 to 7% of its initial value. However, on day 115, when piezometers had to be closed off, it had returned to the same level as in layers 3 and 4, approximately 70% of its initial value, in column R, in contrast, layers 1 to 3 experienced only slight reductions in H C t. Most of the reduction occurred in the top layer (5 to 7 cm from the inlet). This difference in the response of the two columns may have resulted from differences in the way the inocula colonized the sand columns at the beginning of the percolation experiments. Gas accumulation in the gas-trap began much earlier in column R than in column L (see Figure 5). The difference between the two columns progressively decreased, however, and had virtually disappeared by day 130. Shortly thereafter, the gas flow-rate for both columns reached daily values close to or higher than the total pore volume of the sand columns. The proportion of methane in the gas that accumulated in the gas-traps ranged from approx. 20% for column R, when bubbles first formed, to about 75-80% and 85% for columns L
Figure 3. Evidence of desaturation in column L. A bubble is clearly visible in the outlet chamber at the top of the column.
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R (A). and R, respectively, at the end of the experiment. We assume that N z and CO 2 accounted for the rest of the gas. The results presented so far are similar to those described by Reynolds et al. (1992), and strongly indicate that methane gas production by M. barkeri is correlated with the observed decreases in HCsat. As mentioned in the Introduction, a possible mechanism that could bring about these reductions is the blocking of pores by methane bubbles. However, it is not possible on the basis of the information provided so far to determine whether there is a definite causal relationship between methane accumulation and HCsa t decrease. The observed correlation between these two processes may be a by-product of a more fundamental relationship between decreases in HCs~t and some other manifestation of the growth
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D. Sanchez de Lozada et al. and metabolism of M. barkeri 227, e.g. cell proliferation in the pores or production of exopolymers. The results of the biomass assays and the SEM micrographs fortunately allow us to make more definite statements on the mechanisms responsible for the clogging of sand columns. Both columns R and L exhibited a higher biomass density close to the inlet than anywhere else (Figure 6). This observation and the marked H C t reductions in the 0 to 1 cm layer in column L, suggest the existence of a direct relationship between biomass accumulation and clogging, similar to that found by Vandevivere & Baveye (1992a). However, if we use the regression equation of Vandevivere & Baveye (1992a) to obtain a rough estimate of the decreases in H C , to which the highest biomass density observed in column L (1.49 mg wet weight/cm 3 of sand) could lead, we find a relative H C t / HCsat(t=0) value of 90%, much higher than the 3% actually observed. Furthermore, the data for column R do not support the idea of a causal relationship between biomass accumulation and H C t decrease. Indeed, in this column, the most significant biomass accumulation occurred in the first two layers (0 to 1 cm and 1 to 3 cm), the layers that experienced only slight decreases in H C t up to day 115. In addition, although layers 3 (3 to 5 cm) and 4 (5 to 7 cm) had similar biomass densities at the end of the experiment, their hydraulic conducfivifies differed markedly up to day 115, when piezometers had to be closed off. It is possible that between day 115 and the termination of the percolation runs, the conditions changed significantly inside the columns in terms either of HCs~t decreases or biomass density distribution. However, nothing indicates that such changes did occur. The SEM micrographs (Figure 7) show that the cells of M. barkeri were probably distributed randomly in small packets or clumps and not as a uniform biofilm, as some authors have suggested (Okubo & Matsumoto 1979; Taylor et al. 1990). Similar observations were made by Vandevivere & Baveye (1992 a,b). The distribution of these cell clumps appeared very variable in the columns, both horizontally and vertically. For example, one micrograph (Figure 7C) revealed an abundance of clumps while only a few hundred micrometres away, at the same height in column R, no clumps were visible at all (Figure 7D). In all cases, the clumps were very sparse and amounted to only a tiny fraction of the pore space, unlike the distribution of Arthrobacter sp., Bacillus subtilis, and mucoid and nonmucoid variants of a sub-surface isolate observed by Vandevivere & Baveye (1992 a,b,c). Of course, as suggested by Gupta & Swartzendruber (1962), even a small biovolume can be very effective at clogging a porous medium if it is located at strategic points, e.g. at pore necks. However, there is no indication on the micrographs that this was the case in the sand columns. Indeed, most of the cell clumps seemed to be attached to the surfaces of the sand grains. This feature is particularly clear at higher magnification, as in Figure 8A. It is possible that exopolymers were involved in this attachment, although in most instances their presence was not obvious
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(Figures 7 and 8A). However, a few cell clumps were entirely embedded in a dense matrix resembling an exopolymer (Figure 8B). The similar morphology of these mucoid colonies compared to other cells seen indicates that they were not caused by a contaminating organism. In this respect, the strict precautions taken during the experiments to avoid contamination seemed to have been reasonably successful. A few large protozoa of approximately 30 I~m in length were observed at one spot at the inlet surface of column L. No other contaminating organisms were found. The apparently limited spread of the protozoa in column L, indicates that the contamination may have occurred near the end of the experiment, or after stopping the flow of solution. The results described above indicate that methanogens can, under some circumstances, significantlyreduce the hydraulic
Clogging of sand by Methanosarcina barkeri
Figure 7. Scanning electron micrographs of sand grains located within the first mm (A and B) and between 4 and 5 mm (C and D) from the inlet end in column R. Scale bar=100 p.m.
Figure 8. Colonies of Methanosarcina barked 227 attached to sand grains and found between 4 and 5 mm (A) and between 2 and 3 mm (B) from the inlet end in column L. Scale bars= 10 i~m.
World]ournal of Microbiology & 6fotechnology, Vot 10, 1994
~1
D. Sanchez de Lozada et al. conductivity of sand columns. The mechanism responsible for this clogging does not seem to be the accumulation of cells in the pores, nor the production of large quantities of exopolymers. On the basis of the available evidence, we conclude that pore blockage by bubbles of methane produced in situ by the M. barkeri was responsible for the observed decreases in hydraulic conductivity of the sand columns. Reynolds et al. (1992) recently reached a similar conclusion. However, in contrast to the present study, Reynolds et al. (1992) did not rule out any of the alternative mechanisms by which methanogens (or other organisms) could conceivably obstruct pores. The present conclusions should be extrapolated to practical aquifer conditions with extreme caution. At lower temperatures than those of the experiment, or in soils and aquifer materials with very different granulometries and pore geometries, the pattern of pore clogging by methanogens may be quite different from that reported here. In particular, consortia of methanogens or other bacteria involved in interspecific hydrogen transfer may behave very differently to M. barkeri alone, especially in the presence of predators. Further research is needed to assess the effect of these various factors.
Acknowledgements The research reported here was supported in part by a grant from the Subsurface Science Program of the United States Department of Energy. We thank T. Anguish for help in culturing the methanogens and R. Clayton for his contribution to the construction of the permeameters.
References Ahmad, N. 1963 The effect of evolution of gases and reducing conditions in a submerged soil on its subsequent physical status. Tropical Agriculture 40, 205-209. Allison, L.E. 1947 Effect of microorganisms on permeability of soil under prolonged submergence. Soil Science 63, 439-450. Blaut, M., Muller, V., Fiebig, K. & Gottschalk, G. 1985 Sodium ions and an energized membrane required by Methanosarcina barkeri for the oxidation of methanol to the level of formaldehyde. Journal of Bacteriology 164, 95-101. Chang, A.C., Olmstead, W.R., Johanson, J.B. & Yamashita, G. 1974 The sealing mechanism of wastewater ponds. Journal of the Water Pollution Control Federation 46, 1715-1721. Chen, R.L., Keeney, D.R., Konrad, J.G., Holding, A.J. & Graetz, D.A. 1972 Gas production in sediments of Lake Mendota, Wisconsin. Journal of Environmental Quality 1, 155-158. Davis, S., Fairbank, W. & Weisheit, H. 1973 Dairy waste ponds effectively self-sealing. Transactions of the American Society of Agricultural Engineers 16, 69-71. Findlay, R.H., King, G.M. & Watling, L. 1989 Efficacy of phospholipid analysis in determining microbial biomass in sediments. Applied and Environmental Microbiology 55, 28882893.
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Ford, H.W. & Beville, B.C. 1968 Chemical changes in tile-drain filters and ditch banks caused by anaerobiosis. Transactions of the American Society of Agricultural Engineers 11, 41-42. Ghiorse, W.C. & Wilson, J.T. 1988 Microbial ecology of the terrestrial subsurface. Advances in Applied Microbiology 33, 107-172 Gupta, R.P. & Swartzendruber, D. 1962 Flow-associated reduction in the hydraulic conductivity of quartz sand. Soil Science Society of America, Proceedings 26, 6-10. Gupta, R.P. & Swartzendruber, D. 1964 Entrapped air content and hydraulic conductivity of quartz sand during prolonged liquid flow. Soil Science Society of America, Proceedings 28, 9-12. Hilton, J. & Whitehall, K.V. 1979 Investigating reduced hydraulic throughput of sand filters. Effluent and Water Treatment Journal 19, 15-25. Jones W.J., Nagle Jr, D.P. & Whitman, W.B. 1987 Methanogens and the diversity of archaebacteria. Microbiological Reviews 51, 137-177. Jury, W.A., Gardner, W.R. & Gardner W.H. 1991 Soil Physics. New York: John Wiley & Sons. Kristiansen, R. 1981 Sand-filter trenches for purification of septic tank effluent: 1. The clogging mechanism and soil physical environment. Journal of Environmental Quality 10, 353-357. Lance, J.C. & Whisler, F.D. 1972 Nitrogen balance in soil columns intermittently flooded with secondary sewage effluent. Journal of Environmental Quality 1, 180-186. Lee, M.D., Thomas, J.M., Borden, R.C., Bedient, P.B., Wilson, J.T & Ward, C.H. 1988 Biorestoration of aquifers contaminated with organic compounds. CRC Critical Reviews in Environmental Control 18, 29-89. Lobo, A.L. & Zinder, S.H. 1988 Diazotrophy and nitrogenase activity in the archaebacterium Methanosarcina barkeri 227. Applied and Environmental Microbiology 54, 1656-1661. Lovley, D.R. & Phillips, E.J.P. 1987 Competitive mechanisms for inhibition of sulfate reduction and methane production in the zone of ferric iron reduction in sediments. Applied and Environmental Microbiology 53, 2636-2641. Mah, R.A., Smith, M.R. & Baresi, L. 1978 Studies on an acetatefermenting Methanosarcina. Applied and Environmental Microbiology 35, 1174-1184. Oberdorfer, J.A. & Peterson, F.L. 1985 Waste-water injection: geochemical and biogeochemical clogging processes. Ground Water 23, 753-761. Okubo, T. & Matsumoto, J. 1979 Effect of infiltration rate on biological clogging and water quality changes during artificial recharge. Water Resources Research 15, 15361542. Okubo, T. & Matsumoto, J. 1983 Biological clogging of sand and changes of organic constituents during artificial recharge. Water Research 17 813-821. Reynolds, W.D., Brown, D.A. Mathur, S.P. & Overend, R.P. 1992 Effect of in-situ gas accumulation on the hydraulic conductivity of peat. Soil Science 153, 397-408. Ripley, D.P. & Saleem, Z.A. 1973 Clogging in simulated glacial aquifers due to artificial recharge. Water Resources Research 9, 1047-1057. Sanchez de Lozada, D. 1992 Reduction of the hydraulic conductiviW of sand columns by Methanosarcina barkeri 227. MSc. Thesis. Cornell University, Ithaca. Shaw, J.C., Branhill, B., Wardlaw, N.C. & Costerton, J.W. 1985 Bacterial fouling in a model core system. Applied Microbiology 49, 693-701.
Clogging of sand by M e t h a n o s a r c i n a barkeri Stevens, T.O., McKinley, J.P. & Fredrickson, J.K. 1993 Bacteria associated with deep, alkaline, anaerobic groundwaters in southeast Washington. Microbial Ecology 25, 35-50. Swartzendruber, D. & Gupta, R.P. 1964 Possible role of methane in affecting the hydraulic conductivity of fine quartz sand. Soil Science 98, 73-77. Takai, Y., Koyama, T. & Kamura, T. 1956 Microbial metabolism in reduction processes of paddy soils (Part 1). Soil Scienceand Plant Nutrition 2, 63-66. Taylor, S.W., Milly, P.C.D. & Jaffe, P.R. 1990 Biofilm growth and the related changes in the physical properties of a porous medium. 2. Permeability. WaterResourcesResearch26, 2161-2169. Tollner, E.W., Hill, D.T & Busch, C.D. 1983 Manure effects on model lagoons treated with residues for bottom sealing. Transactions of the American Society of Agricultural Engineers 26, 430-435. Van Beek, C.G.E.M. 1984 Restoring well yield in the Netherlands. Journal of the American Water Works Association 76, 66-72. Vandevivere, P. & Baveye, P. 1992a Saturated hydraulic conductivity reduction caused by aerobic bacteria. Soil Science Society of American, Journal 56, 1-13.
Vandevivere, P. & Baveye, P. 1992b Effect of bacterial extracellular polymers on the saturated hydraulic conductivity of sand columns. Applied and Environmental Microbiology 58, 1690-1698. Vandevivere, P. & Baveye, P. 1992c Relationship between transport of bacteria and their clogging efficiency in sand columns. Applied and Environmental Microbiology 58, 2523-2530. Vandevivere, P. & Baveye, P. 1992d Sampling method for the observation of microorganisms in unconsolidated porous media via scanning electron microscopy. Soil Science 153, 482-485. Vandevivere, P. & Baveye, P. 1992e Improved preservation of bacterial exopolymers for scanning electron microscopy. Journal of Microscopy 167, 323-330. Zeikus, J.G. 1977 The biology of methanogenic bacteria. Bacteriological Reviews 41, 514-541.
(Received in revised form 15 October I993; accepted 8 December 1993)
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Decrease of the hydraulic conductivity of sand columns by Methanosarcina barkeri D. Sanchez de Lozada, P. Vandevivere, P. Baveye* and S. Zinder The extent to which a methanogen can clog sand columns was examined: two permeameters packed with clean quartz sand were sterilized, saturated with water, inoculated with Methanosarcina barkeri and percolated under upward flow conditions. After approx. 5 months, the hydraulic conductivity of the sand had decreased to 3% and 25% of the highest values measured earlier. At that point, gas-filled regions in the sand were clearly visible through the transparent walls of the permeameters, and methane bubbles were continuously released from the columns into the effluent. Scanning electron microscopy observations and biomass assays indicated that cell mass accumulation did not contribute significantly to the observed decrease of the hydraulic conductivity. This decrease was therefore attributed to pore blocking due to the entrapment of methane bubbles. Key words: Aquifer materials, clogging, hydraulic conductivity, methane production, methanogenesis, pore blockage.
In the last few decades, the clogging of waterlogged soils and aquifer materials has been the object of a sustained research effort, primarily because of the adverse effects it has on a number of processes, such as the disposal of waste water (Davis et al. 1973; Chang et al. 1974), groundwater recharge (Ripley & Saleem 1973; Oberdorfer & Peterson 1985), groundwater production (Van Beek 1984) or the in situ bioremediation of contaminated aquifer sediments (Lee et al. 1988). The clogging of saturated soils and aquifer materials is manifested by an often drastic decrease in their ability to transmit water, i.e. of their saturated hydraulic conductivity, H C s a t . The various mechanisms suggested to account for this phenomenon are usually classified as physical, chemical or biological. Physical mechanisms include the filtration of solid particles suspended in the percolating liquid, and the progressive disintegration of the soil structure. Chemical phenomena that affect the H C t involve changes in the swelling properties of soils and the dispersion of colloidal particles brought about by variations in the composition of the liquid phase. The mechanisms by which living organisms influence the HCs, t of
O. Sanchez de Lozada and P. Baveye are with the Department of Soil, Crop
and Atmospheric Sciences, Bradfield Hall, Comen University, Ithaca, NY 13853, USA; fax: 607 255 2644. P. Vandevivere is with the College of Marine Studies, University of Delaware, Lewes, DE 19958, USA. S. Zinder is with the Department of Microbiology, Rice Hall, CorneU University, Ithaca, NY 14853, USA. * Corresponding author.
soils and aquifers are by far the least understood. The microflora that are involved in this process consist predominantly of bacteria (Van Beek 1984; Shaw et al. 1985) although fungi (Ripley & Saleem 1973; Okubo & Matsumoto 1983) and protozoa (Hilton & Whitehall 1979) may sometimes be involved. The mechanism most often advanced to account for the observed bacterial reduction of H C t in soils and aquifer materials involves the production of extracellular polymers (Allison 1947; Shaw et al. 1985). Vandevivere & Baveye (1992a) showed, however, that the production of extracellular polymers was not necessary for severe bacterial clogging to occur. Other mechanisms that may come into play and have been invoked occasionally in the literature include plugging of the pores by the bacterial cells themselves (Gupta & Swartzendruber 1962), precipitation in soil pores of iron and magnesium sulphides resulting from the activity of sulphate-reducing bacteria (Ford & Beville 1968; Van Beek 1984), deposition of iron hydroxides or manganese oxides produced by iron bacteria (Van Beek 1984), or the production of gas (Ahmad 1963), in particular by denitrifiers (Lance & Whisler 1972; Oberdorfer & Peterson 1985) and possibly by methanogens (Swartzendruber & Gupta 1964; Gupta & Swartzendruber 1964; Reynolds et al. 1992). To control the extent and intensity of bacterial clogging in sub-surface environments, more detailed information is
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required on the various mechanisms listed above than is currently available. In particular, a fundamental understanding is needed of the mechanisms that could cause the clogging of porous media by methanogens, which are ubiquitous in sub-surface environments (Lovley & Phillips 1987; Ghiorse & Wilson 1988; Stevens et al. 1993). One of the mechanisms by which methanogens could significantly affect HCsa t is via the release of methane. Takai et al. (1956) measured rates of methane production as high as 8 m l / d a y . g soil in a submerged paddy soil under static conditions. On the basis of a methane concentration of 3% ( v / v ) at saturation (Swartzendruber & Gupta 1964), such a production would lead to supersaturation in methane and possible bubble formation when the rate of water transport in the soil was less than approximately 6 x 1 0 -s m / s , a situation that is of widespread occurrence. This observation may explain the fact that the gas phase in ponded softs often contains significant amounts of methane (Kristiansen 1981; Tollner et al. 1983). Once the soil solution has become supersaturated, entrapment of methane bubbles may lead to pore blocking and, consequently, to a reduction of the hydraulic conductivity of the medium. The experimental evidence that is available on this process is still largely inconclusive. On the basis of the results of percolation experiments in sand columns, Swartzendruber & Gupta (1964) concluded that the production of methane by microorganisms could not cause clogging. This statement appears, however, unwarranted in view of the fact that no methanogens were involved in the experiments and that the solutions percolated through the sand were undersaturated with respect to methane, making the formation of bubbles in the pore space very unlikely. More recently, Reynolds et al. (1992) monitored the m o v e m e n t of de-aired, temperature-equilibrated water through repacked laboratory columns of catotelm peat. In unsterilized columns, the hydraulic conductivity decreased sharply over a period of 78 days, while, concomittantly, the volumetric gas content and the gaseous methane concentration in the peat increased significantly. These observations suggest that in some cases there may be a strong correlation between in situ accumulation of anaerobe-generated methane and severe decreases in hydraulic conductivity. However, since Reynolds et al. (1992) did not investigate other possible mechanisms (e.g. biomass accumulation, extracellular polymer production) by which methanogens could clog the porous media, one cannot deduce from the existence of this correlation that there is a causal link between methane production and decreases in hydraulic conductivity. In the present study, laboratory experiments were conducted to appraise the extent to which a methanogen, Methanosarcina barkeri, could decrease the hydraulic conductivity of sand columns. A second objective of these experiments was to elucidate the mechanisms responsible for the observed clogging.
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Materials and Methods Bacterial Strain and Nutrient Solution The bacterial strain used in the experiments was originally isolated from sewage sludge (Mah et al. 1978) and is classified as Methanosarcina barkeri 227, a strict anaerobe that can use a number of substrates: H2/CO2, methanol acetate, formate or methylamines (Blaut et al. 1985; Jones et al. 1987). The deoxygenated solution used to grow the bacterium in batch cultures, which was also percolated through the sand columns, contained (rag/1 deionized water): methanol, 1483; NH4C1, 500; KH2PO4, 740, K2HPO4, 200; MgCI2. 6H20, 100; NAG1, 667; NaHCO 3, 37.5; Na2S.9H20, 281; and CaC12, 45; in addition to 0.875 ml of Resazurin 0.1%/1 and 10 ml of a trace element stock solution/1 (Zeikus 1977; Lobo & Zinder 1988). The phosphate buffer maintained the pH of the solution at 6.8. The preparation method was identical to that adopted by Lobo & Zinder (1988) except for a change in buffer and the fact that the nutrient solution was kept under a pure N 2 atmosphere rather than under a 70% N2/30% CO2 (v/v). Further details on the preparation of the nutrient solution are given by Sanchez de Lozada (1992). Sand The sand used in this study originated from the same site in Arenzville (Cass County, IL, USA) as that used by Vandevivere & Baveye (1992a,b,c). It contains quartz (92%) and magnetite (8%). The particle size range was narrowed down to between 63 and 125 mm by sieving. The sand was washed with sodium acetate (pH 5) to remove carbonates, H202 (6%) to remove organic matter and a mixture of sodium citrate, NaHCO 3 and sodium hydrosulphite to remove iron oxides, autoclaved for 90 min and dried at 105°C. Flow System The flow system is represented schematically in Figure 1. The nutrient solution, held aseptically in 10-1 Pyrex bottles that functioned as Mariotte devices, was percolated under constant hydraulic head conditions in permeameters packed with sand. Two such permeameters were operated simultaneously (columns L and R). Each consisted of an acrylic cylinder (70 mm in length and 25 mm inside diameter) capped at both ends with headpieces manufactured with a conical chamber to ensure an axial flow within the sand and to facilitate the entrainment of gas bubbles. Rubber O-rings provided a good seal at the contact between the acrylic cylinder and the headpieces. The sand was confined within the cylinder by nylon meshes (53-~tm pore size), which in turn were supported by coarser steel meshes. Packing of the sand in the permeameters was achieved by gently tapping the plexiglass cylinder a few times after each addition of a 5 mm layer. Once packed in the column, the sand had a porosity (volume of voids divided by total volume) of 0.39 _+0.01. Except for the columns, which were sterilized by overnight exposure to ethylene oxide, all parts in contact with the flowing solution were autoclaved and assembled aseptically in a laminar-flow hood. The sand was slowly saturated from beneath and a steady upward flow of, autoclaved water was maintained for 7 days, until entrapped air was removed. Each column was then inoculated by injecting, through the septa of the inoculation ports (Figure 1), 10 ml of an exponentially-growing culture of M. barkeri. During the inoculation and shortly thereafter, the flow of solution
Cloggingof sand by Methanosarcina barkeri
Gas-trap N~ N 2
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Figure 1. Schematic diagram of the flow system. to the columns was stopped to foster cell attachment. The flow experiments were conducted at room temperature (20 +_3°C). Since M. barkeri 227 is extremely sensitive to the presence of 02, it was necessary to conduct the percolation experiments under anoxic conditions. To this end, a N2-cylinder and a set of tubes and connectors were used to maintain a N 2 atmosphere in the head space in the nutrient solution bottles and above the water columns in the piezometers (Figure 1). The tubing connecting the solution bottles to the permeameters was made of butyl rubber in order to decrease the diffusion of 02 in the flow system. Samples of the permeameter effluents were taken at 7-day intervals through the sampling ports (Figure 1) to measure changes in the concentration of dissolved methane by GC. The presence of a gas-trap (Figure 1), a piece of glass tubing of known internal diameter terminated by a rubber septum, made it feasible to monitor the volume and composition of the gas bubbles escaping at the top of the sand column. The volume of the gas in the trap was calculated on the basis of the position of the water
meniscus in the glass tube. Once the trap was filled, the gas was evacuated through the spetum with a sterile syringe. Upon initiation of bubble formation, as well as toward the end of the experiments, the relative methane content in the gas was measured by GC. Measurement The hydraulic conductivity (HCsa,) can be calculated from measurements of the hydraulic head gradient using Darcy's equation (Jury et aL 1991) HCsa t
AH
Q=AxHC
tx Az
where Q is the flow rate, A is the cross-sectional area of the sand column, H is the hydraulic head (water potential per unit weight), and Az is the distance between the two points where H
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D. Sanchez de Lozada et al. is measured. The head difference between the two points, AH, is the difference in elevation of water columns held in small tubings, called piezometers, connected to these points at one end and open to the atmosphere (in this case, to a N 2 atmosphere) at the other end. These piezometers, in duplicates at each depth, protruded horizontally about 2 mrn inside the sand columns and were capped with nylon mesh (secured with tygon tubing) to prevent the sand from creeping in. They were inserted at five locations in the permeameters (see Figure 1); just below the inlet mesh, at 10, 20, and 50 nun above the inlet mesh and, finally, above the outlet mesh. Thus HC~t could be calculated for four different layers in the sand columns. In order to facilitate the analysis of the results, H C t values for each column were divided by the initial value (HCsat(t_o),) SO that the conductivity ratio (HCt/HCt(t=o)) was 1 at the time of inoculation. The average value ofHC sat(t-o) was 5.55 x 10-3(± 0.65 x 10-3) cm/s, corresponding to a flux density of approximately 8.2 cm/s. In the remainder of this article, we shall use the notation HCsat for the hydraulic conductivity even after providing evidence that a portion of the sand columns were not completely water-filled during the experiments. The subscript 'sat' should then be interpreted as indicating that the water was under a positive hydraulic head throughout the columns, i.e. was not retained in the sand by capillary action.
Gas Chromatography The analysis of methane in the permeameter effluents and gastraps was performed with a 5890A gas chromatograph (Hewlett Packard), using a 3.1-m Hayesep Q packed column connected to a H 2 flame ionization detector. Nitrogen was used as the carrier gas, flowing at 30 ml/min. The temperature was set at 130°C for the injector, oven and detector. Samples of the gas accumulated in the gas-traps were taken using 1 ml B-D Glaspak tuberculin syringes fitted with Mininert syringe valves, as described in Lobo & Zinder (1988). The same equipment was used for samples from the natural gas supply, used as standards. For the samples of effluent taken from the sampling port, the syringe valve was replaced with sterile 0.2-~tm pore Gleman Acrodisc syringe filters to avoid injection of bacteria into the GC columns.
Biomass Assay and Scanning Electron Microscopy After the flow was stopped at the end of the experiment, each permeameter was dismantled. The sand contained in the plexiglass cylinders was removed in successive layers of 10 mm thickness, after taking samples for scanning electron microscopy (SEM) observations. Using the method developed by Vandevivere & Baveye (1992d), two samples were taken at the inflow side of each of the sections corresponding to the distance intervals 0 to 10, 20 to 30, 40 to 50 and 60 to 70 mm from the column inlet, and were prepared for SEM using a glutaraldehyde/lysine mixture for fixation of the bacterial cells (Vandevivere & Baveye 1992e). Observations were made at three depths in each sample, at approximately 2 mm intervals, starting at the upstream side. The remaining sand from each layer was used to determine the average biomass density. The lipid-bound phosphate (in nmol) of the bacteria in the sand was determined by the assay of Findlay et al. (1989). The number of bacteria per volume of sand (X) was estimated from the resulting data by comparison with the lipid-bound phosphate extracted from a volume of a suspension with a known cell concentration. The mass per cell (Y) was obtained by estimating the average cell volume (on a wet mount under a light microscope), and by multiplying this volume by a
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wet density of 1.1 m g / m m 3, as described in Vandevivere & Baveye (1992a). An estimate of the biomass density was then obtained by taking the product of X and Y.
Results and D i s c u s s i o n The first manifestation of the activity of Methanosarcina barkeri 227 in the columns was a progressive increase in the concentration of methane dissolved in the column effluents (Figure 2). After approximately 100 days of flow for both columns, the methane concentration was within experimental error of the saturation level. Under equilibrium conditions, this would normally have marked the onsert of bubble formation. However, visual evidence suggested that, perhaps as a result of internal mixing, the increase in methane concentration in the effluents lagged behind that occurring in localized regions in the columns. Indeed, on day 89, bubbles started forming in one of the two piezometers situated 10 m m above the inlet mesh in column L. Simultaneously, and at approximately the same level in the column, entrapped gas was clearly visible through the transparent plexiglass walls of the permeameter (see Figure 3). Initially, the regions occupied by entrapped gas were small. However, they tended to coalesce progressively, increasing in size and eventually invading the piezometers which rapidly became inoperative in spite of frequent flushing and had to be closed off (on day 115). Desaturation in column R followed a similar pattern except that it started later (on day 97) and, throughout the experiment, took place predominantly within 20 m m of the outlet end of the sand column. Bubbles also formed in the inlet and outlet chambers of both permeameters. Their appearance coincided with the onset of gas accumulation in the piezometers. The bubbles forming in the inlet chamber rapidly coalesced into one large enough to create a gas interface between the bottom of the sand column and the incoming liquid, eventually preventing flow. Therefore it became necessary to evacuate the bubbles periodically from the inlet chambers of both permeameters. The bubbles in the outlet chambers seemed to form within the wire mesh. They grew progressively until they detached from the mesh and moved toward the brass connectors at the outlet end of the permeameters (see the bubble at the top of Figure 3) where they attached to the plexiglass walls until their diameter increased to such an extent that buoyancy forced them out of the permeameters and into the gas-traps. Soon after the beginning of the percolation experiments, the overall (column-averaged) hydraulic conductivity of the columns began to decrease (Figure 4). After a few weeks, it stabilized at approximately 85% of its original value. Given that methanogenic activity was extremely low during the first 40 days of flow, this initial decrease could not have been used by the bacterial population in the columns and may have been due to settling or rearrangement of the sand particles. However, no direct evidence is available to prove
Clogging of sand by Methanosarcina barkeri
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HCa , had dropped to 3% of its original value. In column R, which was dismantled slightly later (on day 151 ), the H C t was much higher, with a ratio of 0.25. In column L, piezometer readings (data not shown) revealed that the reduction in hydraulic conductivity was most pronounced in the inlet layer (0 to 1 cm). The HCs, t of the second layer (1 to 3) cm decreased rapidly between days 85 and 95 to 7% of its initial value. However, on day 115, when piezometers had to be closed off, it had returned to the same level as in layers 3 and 4, approximately 70% of its initial value, in column R, in contrast, layers 1 to 3 experienced only slight reductions in H C t. Most of the reduction occurred in the top layer (5 to 7 cm from the inlet). This difference in the response of the two columns may have resulted from differences in the way the inocula colonized the sand columns at the beginning of the percolation experiments. Gas accumulation in the gas-trap began much earlier in column R than in column L (see Figure 5). The difference between the two columns progressively decreased, however, and had virtually disappeared by day 130. Shortly thereafter, the gas flow-rate for both columns reached daily values close to or higher than the total pore volume of the sand columns. The proportion of methane in the gas that accumulated in the gas-traps ranged from approx. 20% for column R, when bubbles first formed, to about 75-80% and 85% for columns L
Figure 3. Evidence of desaturation in column L. A bubble is clearly visible in the outlet chamber at the top of the column.
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R (A). and R, respectively, at the end of the experiment. We assume that N z and CO 2 accounted for the rest of the gas. The results presented so far are similar to those described by Reynolds et al. (1992), and strongly indicate that methane gas production by M. barkeri is correlated with the observed decreases in HCsat. As mentioned in the Introduction, a possible mechanism that could bring about these reductions is the blocking of pores by methane bubbles. However, it is not possible on the basis of the information provided so far to determine whether there is a definite causal relationship between methane accumulation and HCsa t decrease. The observed correlation between these two processes may be a by-product of a more fundamental relationship between decreases in HCs~t and some other manifestation of the growth
World Journal of Microbiology & Biotechnology, VoJ 10, 1994
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D. Sanchez de Lozada et al. and metabolism of M. barkeri 227, e.g. cell proliferation in the pores or production of exopolymers. The results of the biomass assays and the SEM micrographs fortunately allow us to make more definite statements on the mechanisms responsible for the clogging of sand columns. Both columns R and L exhibited a higher biomass density close to the inlet than anywhere else (Figure 6). This observation and the marked H C t reductions in the 0 to 1 cm layer in column L, suggest the existence of a direct relationship between biomass accumulation and clogging, similar to that found by Vandevivere & Baveye (1992a). However, if we use the regression equation of Vandevivere & Baveye (1992a) to obtain a rough estimate of the decreases in H C , to which the highest biomass density observed in column L (1.49 mg wet weight/cm 3 of sand) could lead, we find a relative H C t / HCsat(t=0) value of 90%, much higher than the 3% actually observed. Furthermore, the data for column R do not support the idea of a causal relationship between biomass accumulation and H C t decrease. Indeed, in this column, the most significant biomass accumulation occurred in the first two layers (0 to 1 cm and 1 to 3 cm), the layers that experienced only slight decreases in H C t up to day 115. In addition, although layers 3 (3 to 5 cm) and 4 (5 to 7 cm) had similar biomass densities at the end of the experiment, their hydraulic conducfivifies differed markedly up to day 115, when piezometers had to be closed off. It is possible that between day 115 and the termination of the percolation runs, the conditions changed significantly inside the columns in terms either of HCs~t decreases or biomass density distribution. However, nothing indicates that such changes did occur. The SEM micrographs (Figure 7) show that the cells of M. barkeri were probably distributed randomly in small packets or clumps and not as a uniform biofilm, as some authors have suggested (Okubo & Matsumoto 1979; Taylor et al. 1990). Similar observations were made by Vandevivere & Baveye (1992 a,b). The distribution of these cell clumps appeared very variable in the columns, both horizontally and vertically. For example, one micrograph (Figure 7C) revealed an abundance of clumps while only a few hundred micrometres away, at the same height in column R, no clumps were visible at all (Figure 7D). In all cases, the clumps were very sparse and amounted to only a tiny fraction of the pore space, unlike the distribution of Arthrobacter sp., Bacillus subtilis, and mucoid and nonmucoid variants of a sub-surface isolate observed by Vandevivere & Baveye (1992 a,b,c). Of course, as suggested by Gupta & Swartzendruber (1962), even a small biovolume can be very effective at clogging a porous medium if it is located at strategic points, e.g. at pore necks. However, there is no indication on the micrographs that this was the case in the sand columns. Indeed, most of the cell clumps seemed to be attached to the surfaces of the sand grains. This feature is particularly clear at higher magnification, as in Figure 8A. It is possible that exopolymers were involved in this attachment, although in most instances their presence was not obvious
330
World Journal of Microbiology & Biotechnology, Vol 10, 1994
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(Figures 7 and 8A). However, a few cell clumps were entirely embedded in a dense matrix resembling an exopolymer (Figure 8B). The similar morphology of these mucoid colonies compared to other cells seen indicates that they were not caused by a contaminating organism. In this respect, the strict precautions taken during the experiments to avoid contamination seemed to have been reasonably successful. A few large protozoa of approximately 30 I~m in length were observed at one spot at the inlet surface of column L. No other contaminating organisms were found. The apparently limited spread of the protozoa in column L, indicates that the contamination may have occurred near the end of the experiment, or after stopping the flow of solution. The results described above indicate that methanogens can, under some circumstances, significantlyreduce the hydraulic
Clogging of sand by Methanosarcina barkeri
Figure 7. Scanning electron micrographs of sand grains located within the first mm (A and B) and between 4 and 5 mm (C and D) from the inlet end in column R. Scale bar=100 p.m.
Figure 8. Colonies of Methanosarcina barked 227 attached to sand grains and found between 4 and 5 mm (A) and between 2 and 3 mm (B) from the inlet end in column L. Scale bars= 10 i~m.
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D. Sanchez de Lozada et al. conductivity of sand columns. The mechanism responsible for this clogging does not seem to be the accumulation of cells in the pores, nor the production of large quantities of exopolymers. On the basis of the available evidence, we conclude that pore blockage by bubbles of methane produced in situ by the M. barkeri was responsible for the observed decreases in hydraulic conductivity of the sand columns. Reynolds et al. (1992) recently reached a similar conclusion. However, in contrast to the present study, Reynolds et al. (1992) did not rule out any of the alternative mechanisms by which methanogens (or other organisms) could conceivably obstruct pores. The present conclusions should be extrapolated to practical aquifer conditions with extreme caution. At lower temperatures than those of the experiment, or in soils and aquifer materials with very different granulometries and pore geometries, the pattern of pore clogging by methanogens may be quite different from that reported here. In particular, consortia of methanogens or other bacteria involved in interspecific hydrogen transfer may behave very differently to M. barkeri alone, especially in the presence of predators. Further research is needed to assess the effect of these various factors.
Acknowledgements The research reported here was supported in part by a grant from the Subsurface Science Program of the United States Department of Energy. We thank T. Anguish for help in culturing the methanogens and R. Clayton for his contribution to the construction of the permeameters.
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(Received in revised form 15 October I993; accepted 8 December 1993)
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