INTRODUCTION

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Microorganisms are used beneficially in many subsurface engineering operations including microbially enhanced oil recovery, biobarrier establishment, and in situ bioremediation. In the oil industry, exopolymer-producing bacteria have shown great promise to enhance oil recovery by bacterial profile modification (BPM). Here bacteria are injected into the reservoir, are fed for a period of time in order to grow, and form a plug which blocks undesirable flow channels. As a result, the injected water flows through the more-desirable channels containing the oil to push it out a production well. Because of economic and operation considerations, the nutrient supply can only be provided for a finite period of time, and it is important to understand if the bacterial plug will remain effective. After the nutrient supply is terminated, the bacteria will undergo starvation and will be exposed to a wide range of pH values ranging from 1 to 14. The lower pH values occur during acidification processes, while the high pH values occur during caustic flooding conditions. In order to carry out bacterial profile modification in an efficient and cost-effective manner, a fundamental understanding of the effect of solution pH on bacterial viability is required. This paper describes the results of a study on the number of culturable cells (Leuconostoc mesenteroides NRRL B-523) under starvation conditions at different pH values.

Under the proper nutrient conditions, cells form biofilms on solid surfaces, and most of the biological activities are carried out within the biofilm (14, 38). The biofilm is composed of microbial cells and metabolic by-products including extracellular polymers (exopolymers). Biofilm growth in porous media can lead to a reduction in permeability, which results in a decrease in the nutrient supply downstream of the area where the biological operation is being carried out (36). Also, cells near the surface of a porous wall will experience a lack of nutrients owing to mass transfer limitations as the biofilm thickness increases (3). In BPM for oil recovery and biobarrier technology, a permeability zone is selectively plugged to divert the fluid flow or to mitigate the diffusion of hazardous chemicals. In BPM, further nutrient supply must be discontinued after a desired permeability is attained (24). In this case, a lack of nutrients will cause the cells to undergo starvation.

Bacterial starvation affects the performance of the biological activities of cells in in situ bioremediation and oil recovery. Cell death or deactivation caused by starvation will slow down or stop the biological processes. Cell death can also affect cell adhesion and biofilm attachment (2, 16), which may jeopardize the stability of a biofilm. However, microbial cells have always found various ways to survive in adverse environments by forming spores, by cell miniaturization, by cannibalism, etc. (27). Bacterial cell viability is affected by the conditions of the environment during starvation. Parameters that can affect bacterial viability during starvation in natural or engineering applications include temperature, trace minerals, solution pH, metabolic by-products, and chemical biocides (1, 9, 35, 43).

Among the complicated subsurface conditions, the effects of pH and mineral ions on the bacterial starvation were investigated, because solution pH and some trace minerals have been known to play an important role in bacterial metabolism (8, 11, 15). The solution pH can be easily changed due to metabolic by-products and other chemicals present in or injected into the subsurface. Trace minerals are also ubiquitous in the subsurface. While many starvation survival studies have been carried out, most of them have focused on spores, marine bacteria, and gram-negative cells (8, 30, 32). Furthermore, few studies have examined the effects of pH and trace minerals on cell culturability during starvation conditions (37, 42). In this study, the combined effects of pH and trace minerals on cell culturability during starvation were investigated. Various pH values ranging from 3 to 13 were examined in batch reactors with and without trace amounts of metal ions of Mg, Fe, and Mn. These ions have been shown to affect not only cell growth but also cell survival during starvation as shown for Aerobacter aerogenes under starvation conditions (42).

L. mesenteroides NRRL B-523 was used as a model bacterium because of its rapid growth and exopolymer production and its potential use in BPM. The species L. mesenteroides is classified as a member of the lactobacilli, which are gram positive. It is spherical (0.5 to 1.2 µm in diameter), forms long chains during growth, is an oxygen-tolerant anaerobe, and it is known to produce extracellular polysaccharides when fed sucrose (20, 22, 25). The sucrose-induced enzyme dextransucrase breaks down sucrose into glucose and fructose and links the glucose molecules into dextran. The cell growth on sucrose was reported to be virtually the same as the cell growth on the stoichiometrically equivalent mixture of glucose and fructose (25). The kinetics of cell growth and enzyme and exopolymer production have been well studied in our laboratory and can be found elsewhere (19, 25).

	MATERIALS AND METHODS

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Cell growth and starvation media. Batch experiments were carried out to determine how the bacteria respond to carbon source depletion in different pH ranges with and without trace minerals. For the pH effects, different pH values (from 3 to 13) were tested using two separate sets of batch reactors in the presence of trace minerals, one with water-insoluble exopolymers and one without exopolymers. The effects of trace minerals were tested at two different pH values, 7 and 4.5, with and without exopolymers. The trace minerals examined were a combination of Mg, Mn, and Fe. The trace minerals were prepared by individually dissolving MgSO₄ · 7H₂O (12.3 g/ml), FeSO₄ · 7H₂O (1.39 g/ml), and MnSO₄ (0.85 g/ml) in deionized water to make stock solutions. Each stock solution (2 ml) was added to 1 dm³ of phosphate-buffered saline solution. The final concentrations of trace minerals in the growth and starvation media were 12.7 µM for MnSO₄, 10 µM for FeSO4 · 7H₂O, and 100 µM for MgSO₄ · 7H₂O. The medium used to grow the inoculum was a combination of yeast extract (1% by weight), NaCl (0.07 M), NH₄Cl (0.06 M), CH₃COONa (0.06 M), ascorbic acid (0.5 g/dm³), trace minerals, and a carbon source (sucrose or combination of glucose and fructose; 0.1 M) in phosphate-buffered saline solution. The experiments were performed at a constant temperature of 25°C. Batch reactors were shaken slowly (~60 rpm) for 1 min every 4 h during growth and once a day during starvation.

Procedures. Two separate sets of experiments were carried out: one for the effects of pH and the other one for the effects of trace minerals.

(i) Experiments for pH effects with water-insoluble exopolymers. Cells were inoculated into 1,000 ml of growth medium which contained sucrose as a carbon source. At the end of the exponential growth (20 h after inoculation), 500 ml of the suspension was taken and centrifuged (6,000 × g) for 20 min to separate cells and water-insoluble exopolymers from suspension. Cells and water-insoluble exopolymers were collected, washed twice with phosphate-buffered saline solution, and transferred to a 500-ml solution at the predetermined pH value to initiate starvation; pH values used were 3, 4, 5, 6, 7, 8, 10, and 12. Samples (1 ml for culturable-cell measurement and 4 ml for exopolymer analysis) were taken every 4 to 6 h to count culturable cells (CFU) during the first week of starvation, once a day for the second week, and every other day during the remainder of the experiments. The spread plate method was used to measure the number of culturable cells. After successive dilutions of a 1-ml sample in phosphate-buffered saline solution (10 ml), 100 µl of diluted suspension was transferred onto an MRS agar plate. The colonies were counted after 1.5 days of incubation at 30°C. A counter (model ZF; Coulter Electronics Inc.) was also used to determine the total number of cells. All experiments were done in triplicate. The solution pH was monitored every 4 h during the first 2 days of starvation and once in 2 or 3 days for the rest of the experiments. Adjustment of the solution pH was done using 1 N NaOH for basic conditions and 1 N acetic acid for acidic conditions.

(ii) Experiments for pH effects without water-insoluble exopolymers. Cells were inoculated into 1,000 ml of growth medium which contained a combination of glucose and fructose as a carbon source. The rest of the experiments were carried out in the same manner as in the previous paragraph.

(iii) Experiments for trace mineral effects. In order to better observe the effects of trace minerals, a separate set of experiments were carried out at two pH values: 4.5 and 7. Cells were inoculated in two separate growth media; one contained sucrose for insoluble exopolymer production, and one contained a mixture of glucose and fructose. Cells were separated after the growth phase, washed with the buffer solution, transferred to starvation solutions, and measured in the same manner as stated above. All experiments were done in triplicate.

Exopolymer measurements. The extracellular polysaccharide (exopolymer) concentration was measured during both the growth and starvation experiments. Samples (4 ml) were taken from original cultures every 2 h during the exponential-growth phase. During starvation, the samples were taken at the same frequency as stated in the above procedure. The samples were then centrifuged (6,000 × g) for 20 min to separate insoluble materials (cells and insoluble exopolymers) from soluble materials (soluble exopolymers and metabolic by-products). To separate the cells from the insoluble exopolymer, a 10% potassium hydroxide solution was added to dissolve the exopolymer, while keeping the cells intact. The cells and dissolved exopolymer were separated by centrifugation, and the supernatant was saved for insoluble exopolymer precipitation. To precipitate the exopolymers, the supernatants were treated with an excess of 100% ethanol. The precipitate was filtered with a 0.2-µm-pore-size nylon filter (Cole-Parmer), and the filter paper was rinsed with 100% ethanol. Precipitated exopolymers were treated in the following manner. Hydrochloric acid (2 M) was added to the samples to a pH of 1 to 2 in 20-ml borosilicate vials with tightly sealed caps, and the samples were boiled for 3 h in a water bath to break down exopolymers into monomers (glucose molecules). After the boiling, the pH of the sample was adjusted to 6.5 with 10% KOH. This solution was then assayed for glucose using the phenol-sulfuric acid assay (12). Detailed procedures for exopolymer separation and analysis are well described by Jeanes (21).

Dextranase measurements. In addition to the dextran analysis, dextranase analysis was carried out to verify that no dextranase was induced by dextran from L. mesenteroides NRRL B-523 during starvation. Measurements for dextranase were carried out in the following manner. Cells were inoculated with insoluble dextran into a phosphate-buffered saline solution (starvation medium; 500 ml). Samples (1 ml) were taken once in 2 or 3 days for 93 days. Samples were centrifuged, and the supernatants were processed using the method described by Somogyi (40).

	RESULTS

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Effects of pH. Figure 1 shows CFU numbers at different pH values for the sucrose-fed cells. Cells showed the highest viability at pH 5, surviving more than 93 days. An average CFU value below 1 at a specific day indicates no culturable cells. For all of the tested pH values except for pH 5, the CFU values decreased until they became nondetectable during the experiment. The data points for CFU values of 0 are not shown in Fig. 1 and 2 due to the log scale of the y axis. As shown in Fig. 2, cells transferred from the glucose-fructose-fed system showed a trend similar to that for the sucrose-fed system. At high pH values (12 and 13) and at low pH values (3 and 4), cells rapidly became nonculturable, i.e., within 2 days of starvation. Trace minerals were present for all cases.

View larger version (28K): [in this window] [in a new window]   FIG. 1.   pH effects on culturable cell number for sucrose-fed cells.

View larger version (29K): [in this window] [in a new window]   FIG. 2.   pH effects on culturable cell number for glucose-fructose (G+F)-fed cells.

Effects of trace minerals. The culturable cell number (CFU) was determined based on the spread plate method, and the results are shown in Fig. 3 and 4. At pH 4.5, cells became nonculturable within 70 days whether the trace minerals were present or not (Fig. 3). Cells with trace minerals showed a greater ability to remain culturable than those without trace minerals. In contrast to the results at pH 4.5, cells undergoing starvation at pH 7 survived up to 95 days as shown in Fig. 4. Cells from the sucrose-fed system survived better than those from the glucose-fructose-fed system in all cases.

View larger version (26K): [in this window] [in a new window]   FIG. 3.   Trace mineral (traces) effects on culturable cell number at pH 4.5. G+F, glucose-fructose.

View larger version (27K): [in this window] [in a new window]   FIG. 4.   Trace mineral (traces) effects on culturable cell number at pH 7. G+F, glucose-fructose.

Exopolymer analysis. Soluble and insoluble exopolymer concentrations were measured during cell growth and during starvation to examine the effects of exopolymers on cell culturability under different pH conditions. The total amounts of exopolymers (both soluble and insoluble) produced in sucrose-fed and glucose-fructose-fed reactors were 0.37 (±0.02) and 0.46 mg/ml (±0.02), respectively. The exopolymer analysis results are summarized in Table 1. Approximately 80 to 98% of soluble exopolymers were produced in both sucrose- and glucose-fructose-fed cases. About sevenfold more insoluble exopolymer was produced in sucrose-fed cultures than in glucose-fructose-fed cultures. The concentration of insoluble exopolymers in glucose-fructose-fed batches was considered below detection, because the averaged value of the measured concentration was within the experimental error range. As shown in Fig. 5, no change in exopolymer concentration was detected throughout the starvation experiments, which indicates that exopolymer was not consumed by the starving bacteria. Furthermore, no dextranase was detected within the error range during the 93 days of starvation in the presence of insoluble dextran, which supports the lack of dextran degradation by starving bacteria.

View larger version (30K): [in this window] [in a new window]   FIG. 5.   Concentrations of exopolymers during growth and starvation. G+F, glucose-fructose.

	DISCUSSION

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The numbers of culturable cells, at various pH values with and without trace minerals, were measured during bacterial starvation. Cells became nonculturable at different rates depending on pH, the presence of trace minerals, and the presence of exopolymers. The relationship between pH and cell decay rate during starvation was explained using the Michaelis pH function.

The culturability of cells grown on a complex medium supplemented with sucrose was a focus of this study. The medium used for the culturable cell count contained the same components as the cell growth medium. Because dextran production is a major concern, bacterial growth and resuscitation were carried out using sucrose-supplemented media. However, it is possible that L. mesenteroides can enter the viable but nonculturable (VBNC) state under starvation conditions, and as a result, no culturable cells were detected with the resuscitation medium we have used (39). The VBNC phenomenon has been described for over 30 bacterial species (33, 34, 44). Although the VBNC state is still controversial, more studies are necessary to understand the viability of L. mesenteroides under starvation conditions (5, 17, 33).

Effects of pH. The cells were transferred to pH-adjusted phosphate-buffered saline solutions which contained trace minerals. To analyze the cell decay kinetics at different pH values, a first-order decay model was used to describe the culturable population (CFU) measurements: n = n₀ exp(k_dt) where n is the number of CFU per milliliter, n₀ is the initial number of CFU per milliliter, k_d is the decay rate constant (in inverse days), and t is the starvation time (in days). k_d values in Fig. 6 and 7 were determined using linear regression of a plot of the ratio of culturable cells to the maximum number of culturable cells at the beginning of starvation. In the experiment at solution pHs 5 and 6, there were negative deviations from the fitted equation at early time periods and positive deviations at later time periods, which suggests that some fraction of the population is more resistant to starvation at pHs 5 and 6. Some cells were still culturable after long-term starvation periods at pH 5. The k_d values for different pH conditions are summarized in Table 2.

View larger version (27K): [in this window] [in a new window]   FIG. 6.   Proportion of culturable cells during starvation and data fitting with an exponential function for sucrose-fed cases.

View larger version (30K): [in this window] [in a new window]   FIG. 7.   Proportion of culturable cells during starvation and data fitting with an exponential function for glucose-fructose (G+F)-fed cases.

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View larger version (19K): [in this window] [in a new window]   FIG. 8.   Inverse of death rate constant (kd') and model equation as a function of [H+] for sucrose-fed and for glucose-fructose (G+F)-fed cells.

Effects of exopolymers. In the starvation experiments, only the insoluble exopolymers were present with cells, because the growth medium suspension was removed by centrifugation at the end of growth phase, with only the insoluble material (cells and insoluble exopolymers) being transferred to starvation reactors. As shown in Table 1, insoluble exopolymer was a factor of 7 greater in the sucrose-fed batches than in the glucose-fructose-fed batches. As a result, cells transferred from the sucrose-fed culture solutions had more insoluble exopolymers than cells from glucose-fructose-fed batches. The only difference between cells from sucrose-fed and glucose-fructose-fed batches is the presence of insoluble exopolymer, and cells with insoluble exopolymers (i.e., cells from the sucrose-fed reactor) showed better culturability than cells with negligible exopolymer concentration. Consequently, the insoluble exopolymer appears to contribute to the greater culturability of cells during starvation. Exopolymer has been known to help cells store nutrients and to protect cells from a harsh environment (13). As shown in Fig. 6 and 7, in the presence of a high concentration of insoluble exopolymers (sucrose-fed cases), there were a greater number of culturable L. mesenteroides cells in the tested pH range than in the presence of a negligible amount of insoluble exopolymers (glucose-fructose-fed cases). No changes in the exopolymer concentration were observed, indicating that the exopolymer matrix is not degraded by the bacteria undergoing starvation, and this result is promising for BPM to maintain biofilm plugging for long periods of time under starvation conditions.

Effects of trace minerals. The number of culturable cells was normalized based on the maximum number that appeared after starvation began. To determine the decay rate constants, the proportion of culturable cells was fitted with a first-order decay model. The k_d values are summarized in Table 3. At each solution pH, the effect of exopolymers during starvation was also examined. As shown in Table 3, k_d is larger for glucose-fructose-fed cells for both pH values of 4.5 and 7 than for sucrose-fed cells.