APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1994, p. 1481-14860099-2240/94/$04.00+0Copyright ©D 1994, American Society for Microbiology

Effect of Grain Size on Bacterial Penetration, Reproduction,and Metabolic Activity in Porous Glass Bead Chambers

PRAMOD K. SHARMAt AND MICHAEL J. McINERNEY*Department of Botany and Microbiology, University of Oklahoma, Nonnan, Oklahoma 73019-0245

Received 13 September 1993/Accepted 15 February 1994

We determined the elfects of grain size and nutritional conditions on the penetration rate and metabolicactivity ofEscherichia coli strains in anaerobic, nutrient-saturated chambers packed with different sizes of glassbeads (diameters, 116 to 767 jum) under static conditions. The chambers had nearly equal porosities (38%) butdifferent calculated pore sizes (range, 10 to 65 ,um). Motile strains always penetrated faster than nonmotilestrains, and nutrient conditions that resulted in faster growth rates (fermentative conditions versus

nitrate-respiring conditions) resulted in faster penetration rates for both motile and nonmotile strains for allof the bead sizes tested. The penetration rate of nonmotile strains increased linearly when bead size was

increased, while the penetration rate of motile strains became independent of the bead size when beads havingdiameters of 398 ,um or greater were used. The rate of H2 production and the final amount of H2 produceddecreased when bead size was decreased. However, the final protein concentrations were similar in chamberspacked with 116-, 192-, and 281-jim beads and were only slightly higher in chambers packed with 398- and767-jim beads. Our data indicated that conditions that favored faster growth rates also resulted in fasterpenetration times and that the lower penetration rates observed in chambers packed with small beads were dueto restriction of bacterial activity in the small pores. The large increases in the final amount of hydrogenproduced without corresponding increases in the final amount of protein made indicated that metabolismbecame uncoupled from cell mass biosynthesis as bead size increased, suggesting that pore size influenced theefficiency of substrate utilization.

Recent studies have clearly shown that many deep subsur-face environments like the Atlantic Coastal Plain sedimentspossess climax ecological communities characterized by a highdegree of microbiological diversity, a trophic structure, mate-rials cycling, and energy transfer (3, 5, 11, 12, 20, 28, 30, 31, 36).The factors that control the movement, activity, and distribu-tion of microorganisms in deep subsurface environments arenot well understood. In many unconsolidated aquifers, adsorp-tion is the dominant factor controlling microbial transport (10,13, 33). However, straining becomes an important removalmechanism when the average cell size is greater than the sizesof 5% of the grains that compose the porous material (14, 17,38). Unconsolidated sediments composed of fine sands, silts,and clays have effective pore sizes that are within the size rangeof most bacterial cells (23), suggesting that straining may be amajor factor restricting the transport of bacteria through thesematerials. This should also be true for consolidated rockformations (9). Jenneman et al. observed that the rate ofpenetration of a Bacillus strain was very slow in sandstonecores with permeabilities less than 100 mDarcies, presumablybecause the large proportion of small pores restricted bacterialmovement (18, 19). These observations suggest that the grainsize of porous materials may be an important factor governingmicrobial movement.

Small pore sizes have been shown to influence microbialgrowth and activity. Several studies have shown that microbialcell division is restricted in rectangular capillaries with smallcross-sectional areas (20 to 50 ,um) and that this cannot beattributed to nutrient limitation or a lack of oxygen (27, 40, 41).

* Corresponding author. Mailing address: Department of Botanyand Microbiology, University of Oklahoma, 770 Van Vleet Oval,Norman, OK 73019-0245. Phone: (405) 325-6050.

t Present address: Department of Civil Engineering, Stanford Uni-versity, Stanford, CA 94305-4020.

Sharma et al. found that the rate of growth of Escherichia coliwas slower, and the lag time was longer, in sand-packedchambers than in liquid culture, again suggesting that smallpore spaces restrict bacterial cell division (35). Jones et al. (20)found that the number of sulfate-reducing bacteria in deepsubsurface sediments was inversely correlated with the claycontent. Also, the rate of [2-14C]acetate and [U-'4C]glucoseoxidation was faster in the anaerobic sandy sediments of a deepaquifer than in the clayey sediments of the confining layers (5).In these clayey sediments, more than 80% of the pore throatshad diameters of 0.5 ,um or less, and 50% of the pore throatsin the sandy sediments had pore diameters greater than 5 jim

(5). The lower activity in the clayey sediments was attributed tothe large proportion of small pores; however, other factors,such as hydraulic conductivity and organic carbon content, mayalso have affected microbial activity.

In this study, we examined the effect of grain size on thepenetration rate and activity of E. coli by using a model porousglass bead system. The porosities and pore volumes of differentporous chambers were nearly identical regardless of the beadsize used, and little or no variation in permeability wasobserved in replicate chambers packed with beads of the samesize. Thus, the amounts of nutrients available for bacterialproduction were identical in all chambers, and since thepermeabilities of replicate chambers did not differ, the cham-bers provided an ideal system to study the effects of animportant hydrogeological factor, grain size, on microbialpenetration, cell mass biosynthesis, and metabolic activity.Since it has been suggested that directional self-locomotiveresponses of bacteria to certain stimuli are an important modeof transport in aquifers with low flow rates and are importantin the dispersal of microorganisms away from the dominantflow channels (15, 24), we also studied the impact of differentbiological factors, such as motility, gas production, and thenutritional status of the medium, on microbial penetration

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through chambers packed with different sizes of beads understatic conditions. Our approach for understanding how a

specific cellular process influences bacterial penetration was tocompare the penetration rates of mutant strains of E. coli withthe penetration rates of the respective isogenic parentalstrains. While E. coli is not a subsurface microorganism, theresults of our experiments clearly illustrated how the loss of a

genetically defined function affects bacterial penetration.

MATERIALS AND METHODS

Bacterial strains and growth conditions. The four E. colistrains which we used were obtained from A. Bock, and thephenotypic and genotypic characteristics of these organismshave been described previously (32). For experiments con-ducted under fermentative conditions, all of the strains were

grown anaerobically in motility growth medium (MGM) (pH7.0) (32, 35), which was a mineral medium supplemented withgalactose, peptone, and L-methionine. For nitrate-respiringconditions, the strains were grown in galactose-nitrate me-

dium, which was similar to MGM except that it lacked peptoneand sodium nitrate (0.3%, wt/vol) was added. Cultures were

incubated at 35°C under static conditions. All strains were

maintained on nutrient agar (2%, wt/vol) slants. Cultures were

transferred to fresh slants every 15 days. The purity of cultureswas routinely checked microscopically and by determiningGram reactions. The motility of each strain was determined byusing MIO broth (Difco Laboratories) and by microscopicexamination.Anaerobic media and solutions were prepared and used as

described previously (2, 4). The gas phase used for all anaer-

obically prepared media and solutions was the gas phase of an

anaerobic chamber (about 1 to 5% H2, with the balance N2).The specific anaerobic growth rate at 23°C for each strain ineach medium was determined by measuring changes in A600(25).Porous medium experiments. Five different sizes of acid-

washed glass beads (Sigma Chemical Co., St. Louis, Mo.) were

used as the porous material. Glass chambers were prepared,and each chamber was packed with one size of glass beads as

described previously (32).To determine how grain size affects the movement of E. coli

strains, duplicate, sterile, nutrient-saturated, 10-cm-long cham-bers containing beads of a particular size were placed in a

horizontal position inside the anaerobic chamber, and eachchamber containing beads was inoculated with 0.1 ml of an

exponentially growing culture (about 107 cells) (35). A syringecontaining the appropriate sterile growth medium was insertedthrough the distal end of the chamber. At hourly intervals, thepresence of viable cells in the syringe at the distal end was

determined by removing the syringe and aseptically inoculating0.1 ml of the syringe contents into a sterile tube containing 3 mlof the medium used to saturate the chamber. A new sterilesyringe filled with the appropriate growth medium was theninserted into the distal end of the core for the next sample. Thetubes inoculated with the contents of the distal syringes were

incubated aerobically at 35°C and examined for the presence ofgrowth. The tubes in which growth was observed were checkedfor purity microscopically and by plating cultures onto platecount agar. Each chamber was monitored for 1,000 h afterinoculation.The penetration time for a chamber was defined as the

number of hours between the time when the chamber was

inoculated and the time when viable cells were detected in thedistal syringe. The penetration rate was calculated by dividingthe length of the core (in centimeters) by the penetration time.

In each experiment, the pore volume (in milliliters), theporosity (expressed as a percentage of the total volume), andthe permeability (in Darcies) of each chamber were deter-mined as described previously (19). The pore size for closelypacked glass spheres was calculated from the following equa-tion, as described by Krone et al. (21): pore size (in microme-ters) = 0.085 x diameter of spheres (in micrometers).The average diameter for each size of glass beads was

empirically determined by phase-contrast microscopy (7).Bacterial activity. The effect of grain size on metabolism was

determined by monitoring the gas pressure due to H2 produc-tion inside the chambers. Duplicate chambers were packedwith each size of beads, and each chamber was saturated withMGM and inoculated with 0.1 ml of an exponentially growingculture of strain RW262 (about 107 cells). Chambers that werefilled with MGM but did not contain glass beads served aspositive controls.The gas pressure in each packed chamber was monitored by

the method described by DeWeered et al. (8), except that thetransducer system was modified to provide a 1-ml gas head-space between the chamber and the pressure transducer. Thepressure transducer (series PX136; Omega Engineering, Inc.,Stamford, Conn.) was attached to a 1-ml syringe, and the jointwas sealed with a coating of epoxy to make the apparatusleak-free. An 18-gauge needle was attached to the other end ofthe syringe. The tip of the needle was bent manually at anangle of about 200 to prevent coring of the stopper andplugging of the needle by the glass beads. The needle wasflushed with 100% N2 and then was sterilized with ethyl alcoholjust before it was used. The needle was aseptically inserteduntil the tip of the needle was in contact with the porousmaterial. Each chamber was placed in a vertical position in atest tube rack and was incubated at 37°C for 100 h.The headspace gas pressure was monitored electronically

every 24 or 60 min by a computer-controlled device (8). Thepressure transducers used for the experiment had an electricaloutput that was proportional to the gas pressure in thechamber (1 mV/kPa). The transducer response (in millivolts)to H2 injected into a serum bottle was linear up to anoverpressure of 100 kPa. The full-scale output of each of thetransducers used was about 103 mV. The changes in transduceroutput were processed through a switching circuit and thedigital-analog input-output module of the computer. Thecomputer was used to start the time schedule for recording theelectrical output from each transducer and to collect the data.For each chamber, the amount of H2 produced (in micro-moles) was determined by converting the gas pressure mea-surements into micromoles of H2 by using the ideal gas law(26) and the amount of H2 present in the headspace of thechamber, as determined with a reduction gas analyzer (modelRGA3; Trace Analytical, Menlo Park, Calif.) (22).The amounts of cellular protein in the chambers were

determined after 1,100 h of incubation to ensure that maximalbacterial production had occurred throughout each chamber.The contents of each chamber were transferred to a tubecontaining 4 ml of 20 mM potassium phosphate buffer (pH7.0). Each tube was vortexed for about 1 min to remove thecells from the glass bead matrix. After the solid phase hadsettled in the tube, the supernatant was collected and centri-fuged at 10,000 x g at 4°C for 10 min. The resulting cell pelletwas resuspended in 1 ml of 1 N NaOH and then added to thetube containing the glass beads from which the liquid had beenremoved. The alkali-bead slurry was vortexed for 1 min todistribute the NaOH uniformly, and the tube was then boiledfor 10 min to digest the cells. The amount of protein in eachslurry was determined by the bicinchoninic acid method (37).

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TABLE 1. Physical and hydrodynamic characteristics of chamberspacked with different sizes of glass beadsa

Bead size Pore vol Porosity Permeability Calculated(pm) (ml) (%) (Darcies) pore size

116.0 ± 12.0 7.8 (0.4) 39.0 0.05 ± 0.01 10.0192.0 ± 36.0 7.4 (0.2) 37.0 0.36 ± 0.29 16.0281.0 ± 28.0 7.5 (0.0) 37.5 0.91 ± 1.39 24.0398.0 ± 11.0 7.7 (0.0) 38.5 8.00 ± 2.40 34.0767.0 ± 105.0 7.7 (0.4) 38.5 12.70 ± 2.34 65.0

a The bead size values are means ± standard deviations obtained by measur-ing 10 individual beads of each size. The permeability values are means +standard deviations calculated by using eight chambers packed with each beadsize. The pore volume values are means; the values in parentheses are ranges.The porosity values were determined by using the pore volume values, and thepore sizes were determined from the bead size values.

Statistical analysis. We determined the significance of dif-ferences between the rates of penetration through chamberspacked with different sizes of glass beads for motile andnonmotile gas-producing strains and their isogenic, non-gas-producing derivatives under fermentative or nitrate-respiringconditions by performing a t test (paired comparison) (16). Alinear regression analysis was used to determine whether thepore volumes or the amounts of cellular protein in chambersvaried significantly when chambers containing glass beads ofdifferent sizes were used. A t test was used to determinewhether the slope of the regression line (B) was significantlydifferent from zero (HO: B = 0) (16).

RESULTS

Characteristics of the porous medium. Five different glassbead sizes (diameters, 116 to 767 p.m) were used. The physicaland hydrodynamic properties of core chambers packed witheach size of glass beads are shown in Table 1. Linear regressionanalysis showed that the pore volumes and, consequently, theporosities of chambers packed with glass beads of differentsizes did not vary significantly (P < 0.005). The average porevolume and the average porosity for all of the chambers were7.6 ml and 38%, respectively. The permeability ranged from0.05 Darcies for chambers packed with the smallest beads to12.7 Darcies for chambers packed with the largest beads. Asigmoidal relationship between permeability and bead size wasobserved (data not shown). The calculated pore size rangedfrom 10 p.m for the smallest beads to 65 p.m for the largestbeads.

Factors affecting bacterial penetration. We examined theimportance of growth rate, motility, and gas production formicrobial penetration through porous materials consisting ofdifferent sizes of beads by making paired comparisons betweenthe penetration rates obtained with four E. coli strains (Table2). We observed higher penetration rates with motile strainRW262 and its isogenic non-gas-producing derivative,FM2629, and with nonmotile strain MC4100 and its non-gas-producing derivative, FM909, under fermentative conditions(MGM) than under nitrate-respiring conditions (galactose-nitrate medium) (P < 0.05). The only exception was thepenetration of the two motile strains through chambers packedwith 281-p.m beads. These data support the hypothesis thatconditions which favor more rapid growth of an organism alsofavor more rapid microbial penetration since the growth rateof each strain was greater under fermentative conditions thanunder nitrate-respiring conditions (Table 2).The two motile strains always penetrated chambers faster

TABLE 2. Effect of bead size on the penetration rate of E. colistrains through chambers packed with different sizes of beads

under fermentative and nitrate-respiring conditions

Penetration rate (cm/h)bBead

Strain' Characteristics size Fermentative Nitrate-(pFm)conditions respiring

conditions

RW262 Motile, gas 116 0.032 (0.002) NPcproduced 192 0.211 (0.030) 0.033 (0.014)

281 0.355 (0.000) 0.357 (0.086)398 0.523 (0.000) 0.357 (0.086)767 0.523 (0.000) 0.357 (0.086

FM2629 Motile, no gas 116 0.027 (0.010) NPproduced 192 0.170 (0.052) 0.025 (0.000)

281 0.355 (0.000) 0.418 (0.208)398 0.523 (0.000) 0.400 (0.000)767 0.523 (0.000) 0.357 (0.000)

MC4100 Nonmotile, gas 116 0.018 (0.000) NPproduced 192 0.029 (0.002) NP

281 0.056 (0.006) NP398 0.070 (0.020) 0.064 (0.002)767 0.133 (0.022) 0.089 (0.004)

FM909 Nonmotile, no gas 116 0.016 (0.002) NPproduced 192 0.035 (0.014) NP

281 0.056 (0.006) NP398 0.064 (0.008) 0.045 (0.002)767 0.144 (0.000) 0.073 (0.006)

The specific growth rates of strains RW262, FM2629, MC4100, and FM909under fermentative conditions were 0.66, 0.57, 0.58, and 0.46 h- 1, respectively,while the specific growth rates of these strains under nitrate-respiring conditionswere 0.35, 0.28, 0.35, and 0.35 h- 1, respectively.

b The values are means (and ranges) of data from duplicate chambers. Thechambers were saturated with MGM (fermentative conditions) or galactose-nitrate medium (nitrate-respiring conditions).

' NP, no penetration of cells occurred after 1,000 h of incubation (penetrationrate, less than 0.011 cm/h).

than the nonmotile strains when the bead size was the same(Table 2). The two motile strains also penetrated chamberspacked with 192- or 281-p.m beads under nitrate-respiringconditions, while the two nonmotile strains did not. Althougha direct comparison between motile and nonmotile strainscould not be made because of possible differences in thegenetic backgrounds of the organisms, our data strongly sug-gest that motility was an important factor for penetration. Nosignificant difference (P < 0.05) was observed with any of thebead sizes used between the penetration rates of the gas-producing and non-gas-producing strains under nitrate-respir-ing conditions or between the penetration rates of the twononmotile strains under fermentative conditions. The penetra-tion rates of the two motile strains (a gas-producing strain anda non-gas-producing strain) through chambers packed with116- or 192-p.m beads were significantly different (P > 0.05);however, the difference was very small. From these data, weconcluded that gas production was not an important factorcontrolling penetration through the porous chambers used inthis study.The rate of penetration of each strain decreased when

smaller beads were used regardless of the nutrient conditions(Table 2 and Fig. 1). The relationship between penetrationrate and bead size under fermentative conditions depended onwhether the strain was motile or nonmotile. Figure 1 shows therelationship for motile strain RW262 and nonmotile strainMC4100. The relationship between penetration rate and beadsize for the two non-gas-producing strains (FM2629 andFM909) was similar to the relationship for the respectivegas-producing strains (data not shown). The penetration rate

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1484 SHARMA AND McINERNEY

U-

4)--

E

0

a)

0.5 -

0.4-

0.3 1

o0.

0.1

0 150 300 450 600 750

Bead size (jpm)FIG. 1. Effect of the bead size on the penetration rate of motile

strain RW262 (0) and nonmotile strain MC4100 (0) under fermen-tative conditions. The data were obtained from Table 2.

of nonmotile strain MC4100 increased linearly with bead size(from 0.02 to 0.13 cm/h for chambers packed with 116- and767-p.m beads, respectively). The penetration rate of motilestrain RW262 also increased linearly with bead size for cham-bers packed with 116-, 192-, and 281-pm beads, but thepenetration rate was independent of bead size in chamberspacked with 398- or 767-p.m beads. The penetration rates ofthe two motile strains (RW262 and FM2629) through cham-bers packed with 281-, 398-, or 767-p.m beads under nitrate-respiring conditions were not significantly different (Table 2),again suggesting that penetration rate was independent ofbead size in chambers packed with the larger beads.

Effect of bead size on bacterial activity. Bead size did affectthe extent and rate of hydrogen production by strain RW262grown under fermentative conditions in chambers packed withdifferent sizes of beads (Fig. 2 and Table 3). The rates ofhydrogen production in chambers packed with the two largestbead sizes were about fivefold less than the rates observed incontrol chambers without beads. In chambers packed with116-, 192-, or 281-p.m beads, the rates of hydrogen productionwere 50% lower than the rates observed in chambers packed

1C

E

U)a)0)

0 5 10 15 20 25 30 35 40 45

Time (h)

FIG. 2. Effect of the bead size on in situ metabolism of strainRW262 under fermentative conditions in chambers packed with 116-or 767-[Lm beads and a control chamber filled with liquid medium(MGM) without beads. The data are from a representative chamberfor each bead size or from the control. Pressure was monitored for 100h of incubation. No further increase in pressure was observed after themaximum pressure was reached.

TABLE 3. Effect of bead size on in situ metabolic activity of E. coliRW262 under fermentative conditions

Bead size Cellular protein Rate of H2 Total amt of Time that H2(p'm) concn ([.gIml) production H2 produced production

(Rmol/h) (p.mol) ceased (h)a116 360.0 (34.0)" 0.450 (0.100)" 3.93 (0.00)" 17.0 (2.0)"192 374.0 (96.0) 0.508 (0.220) 9.82 (7.84) 30.0 (20.0)281 355.0 (62.0) 0.526 (0.158) 20.63 (1.96) 55.0 (30.0)398 512.0 (44.0) 1.195 (0.590) 33.39 (5.90) 42.0 (4.0)767 470.0 (34.0) 1.295 (0.206) 34.38 (3.92) 42.0 (4.0)No beads NDC' 6.650 (2.420) 43.20 (0.00) 24.5 (9.0)

"The values include an apparent lag time of 10 h for each type of chamber.^ The values are means (and ranges) of data from duplicate chambers.C ND, not determined.

with the two largest bead sizes. Also, metabolism, as indicatedby the total amount of H2 produced, was markedly inhibited.The amount of H2 produced in chambers packed with 116-p.mbeads was about 10-fold less than the amount produced inchambers packed with 767-p.m beads. The increase in gaspressure ceased after 17 h in chambers packed with 116-p.mbeads and after between 42 and 55 h in chambers packed with281-pm or larger beads. The gas pressure remained unchangedafter extended incubation (100 h), even though viable cellswere present for at least 300 h. The final amounts of proteindetected in chambers packed with 116-, 192-, or 281-p.m beadswere about 23 to 30% less than the final amounts observed inchambers packed with 398- or 767-p.m beads.

Interestingly, the numbers of cells that were produced, asindicated by the final protein concentrations, were similarregardless of the bead size used. Although slightly less protein(23 to 30% less) was detected in chambers packed with 116-,192-, and 281-p.m beads (Table 3), we observed no significantdifferences in the final protein concentrations detected in thechambers.

DISCUSSIONAll of the chambers filled with glass beads had nearly

identical porosities and pore volumes. Thus, the differences ingrowth, activity, or penetration rate which we observed werenot due to differences in the amounts of nutrients present inthe chambers. A constant value for porosity was expected sincethe diameter of a spherical porous material affects pore sizeand, consequently, permeability but not the porosity (6). Onecomplication with our experimental system is that both poresize and surface area change when different sizes of beads areused (29). However, an extensive analysis of the literature onthe influences of surfaces on microbial activity led van Loos-drecht et al. (39) to conclude that there is no experimental ortheoretical evidence that interfaces directly influence microbialactivity. On the basis of our results, we concluded that theobserved effects on the activity and penetration of E. colistrains reported above were probably due to differences in poresize rather than differences in surface area. The effect of poresize on microbial activity is probably an indirect effect becauseof the impaired ability of bacteria to move through small poresand the reduced diffusivity of nutrients and metabolitesthrough the materials. Zvyagintsev (40) noted that little or noBrownian movement of cells occurred in small capillaries(cross-sectional area, 20 p.m2), which suggests that diffusionwas limited. The explanations proposed for the reduced den-sity and activity of aerobic and anaerobic bacteria observed inclayey deep subsurface samples from the Atlantic Coastal Plainsediments of South Carolina include lower pH values (36),

.00 767 m

0 116 m 0sr 030 * Control 0t0M,0O-0_ .

30 6

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1-11-

8

6

4

2

ROLE OF GRAIN SIZE 1485

reduced hydraulic conductivity (12), and small pore throats (5).The results reported in this paper are consistent with thehypothesis that small pore throats restrict microbial activityand penetration.Our results suggest that decreasing the pore size did not

severely inhibit microbial cell division since the final proteinconcentrations in chambers containing different sizes of beadswere similar (Table 3). We have also found that the finalnumber of viable cells of Enterobacter aerogenes remainednearly constant (about 1010 cells) in chambers packed withbeads of different sizes (34). Workers who studied the growthof bacteria and yeasts in rectangular capillaries with smallcross-sectional areas found that the ratios of the final numberof cells to the initial number of cells were similar in smallrectangular capillaries with cross-sectional areas of 20 and 50.m2 , areas which are similar to the pore sizes of the chambers

used in this study. However, when capillaries with largercross-sectional areas were used, much larger ratios wereobserved, indicating that larger pores (>50 RMm2) did notrestrict cell reproduction (27, 40, 41).Ou and Alexander (29) found that Bacillus megaterium

consumed more oxygen, utilized glucose at a faster rate, andwas able to maintain oxygen uptake for longer periods of timewhen it was grown in media in the presence of smaller glassbeads (diameters, 29 and 53 ,um) than when it was grown in thepresence of larger beads or in bead-free medium. We foundthat the activity of E. coli under fermentative conditions wasinhibited in porous materials compared with the activity inliquid culture, and this inhibition was more pronounced asbead size decreased (Table 3 and Fig. 2). This finding isconsistent with the decrease in the diffusivity of nutrients andcells expected as the pore size of a matrix decreases. In thestudy of Ou and Alexander (29), the stimulatory effect ob-served when small beads were used may have been an indirecteffect on the pH of the medium, since the pH in flaskscontaining smaller beads did not decrease as much as the pHin flasks containing larger beads or the pH in flasks withoutbeads (29).There are several possible explanations for the reduced

activity observed in chambers packed with smaller beads. Oneexplanation is that bacterial activity was not inhibited, but thelarge surface area in chambers packed with small beads alteredthe metabolism of the cells so that hydrogen was no longerproduced (39). However, other studies performed with thesestrains of E. coli and with Enterobacter aerogenes, Clostridiumacetobutylicum, and a halophilic anaerobe revealed that thenature of the end products produced did not change when theorganisms were grown in the presence of glass beads, fine sand,or crushed sandstone as the porous material compared withliquid cultures (34, 35). A more likely explanation is that sincecells were inoculated at only one end of the chamber ratherthan being evenly distributed throughout the chamber, thesmall pores restricted the movement of nutrients to the cells orthe movement of cells to the nutrient-rich regions of thechambers. If the rate of diffusion of galactose in the chamberswas similar to the rate of diffusion found for glucose in smallcapillaries (about 0.15 cm/h) (1), about 66 h would have beenrequired for galactose to diffuse from one end of each 10-cm-long chamber to the other. If the cells completely consumed allof the locally available galactose near the point of inoculation,this rate of galactose diffusion was slow enough to affect therate of hydrogen production in chambers packed with smallbeads (Fig. 2), given the slow rate of cell penetration observedthese chambers (Table 2). This does not explain why the totalamount of hydrogen produced was so low in chambers packedwith small beads, since we expected metabolism to continue,

albeit very slowly, in these chambers. No further increase inpressure was observed in these chambers even after 100 h ofincubation (data not shown). It may be that a localizedaccumulation of end products combined with some type ofnutrient starvation may have created an inhibitory microenvi-ronment in which metabolism was inhibited or cell viability waslost before the cells were able to move into other regions of thechambers.The fact that there was little or no difference in the final

amounts of protein but there were large differences in the totalamounts of H2 produced between chambers packed with thesmaller beads and chambers packed with the larger beads(Table 3) suggests that bead size and, consequently pore sizeaffected the efficiency by which E. coli used the substrate. Themost efficient cell mass biosynthesis, based on the amount ofprotein made per hydrogen produced, seems to have occurredin chambers packed with the smallest beads (Table 3). As beadsize increased, the amount of hydrogen produced relative tothe amount of protein increased, suggesting that metabolismbecame increasingly uncoupled from cell mass biosynthesis asbead size increased. However, possible complicating factors,such as localization of the inoculum at one end of the chamber,a shift in metabolism, and inhibitory effects of end products(see above) prevented definitive conclusions from being madeat this time.

Previously, it has been shown that growth rate, motility, andgas production are important factors that affect the penetra-tion rate of E. coli through sand-packed chambers under staticconditions (32). In this work we found that conditions thatsupport faster growth rates (fermentative conditions versusnitrate-respiring conditions) and motility are important factorsthat affect the penetration rate of cells regardless of the poresize of the material. However, paired comparisons between thepenetration rates of motile and nonmotile cells and thepenetration rates of the respective isogenic, non-H2-producingstrains showed that H2 production did not play a major role inthe differences in penetration rates observed under differentmetabolic conditions (Table 2). It may be that in moreheterogeneous porous materials, such as sand, gas productionis important for the penetration of nonmotile strains. Table 2and Fig. 1 conclusively show that bead size affected thepenetration rate and thus is an important physical factordetermining the dispersal and distribution of cells in porousmaterials. For the motile strains, the penetration rates inchambers packed with the two larger bead sizes were similar(Fig. 1), suggesting that pore sizes greater than 34 pum did notrestrict penetration and that the cells were able to penetrate ata rate dictated by their innate biological properties. Thisobservation is consistent with the results of a study on theeffect of permeability on the penetration rate of a halotolerant,motile bacterium, strain BCl-INS, through sandstone coresunder static conditions, in which it was found that the rate ofpenetration of strain BCl-1NS was independent of permeabil-ity in sandstone cores with permeabilities greater than 100mDarcies (19). The results of this and previous studies (18, 19,24, 32) show that bacteria have the ability to penetrate bothconsolidated and unconsolidated porous materials in the ab-sence of fluid flow at ecologically important rates and thatbiological factors, such as growth rate and motility, andphysical factors, such as pore size, may be important factorsgoverning the penetration and distribution of microorganismsin the subsurface.

ACKNOWLEDGMENTSWe thank A. Bock of the Institut fur Genetik und Mikrobiologie,

Munich, Germany, for the bacterial strains, F. Concannon for assis-

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1486 SHARMA AND McINERNEY

tance with hydrogen measurements, R. M. Knapp for verifying themathematical equation used to estimate pore sizes, and P. J. Reynoldsfor assistance with the penetration experiments.

This work was supported by contracts DE-AC22-90BC14662 andDE-FG05-89ER14003 from the U.S. Department of Energy.

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