The effects of soil moisture on microbial reproductive patterns

 

Tia Orr

10/31/07

 

Abstract.  Reproduction patterns are essential to understanding the overall fitness of an organism. K-selected reproductive patterns often occur when conditions are favorable.  r-Selected reproductive patterns often occur when conditions are unfavorable.  Higher moisture in the soil is most favorable for soil microbes. This study aimed to determine if r-Selected and K-selected patterns are indicative of the amount of moisture in the soil.  Soil samples from wetland and grassland from Havenwoods State Forest were collected, cultivated, and incubated at 25 degrees Celsius for three weeks.  Microbes from grassland soil samples grew significantly faster than microbes from wetland samples (p<1.2 x 10-6).  These results imply soil moisture may influence reproductive patterns in soil microbes, though more research is needed to confirm these inferences.

 

 

            Reproductive patterns are an important variable in population dynamics.  Knowledge of how an organism reproduces gives insight into their competitive ability which gives light to their overall fitness.  The environment an organism inhabits plays a significant role in the manner in which the organism reproduces due to biotic and abiotic limitations. 

Organisms reproduce with a K-selected or an r-selected reproductive pattern.  K-selected reproductive patterns tend to occur in environments that are most stable for an organism, providing a sufficient amount of food, resources, and habitat for the more competitive individuals in the species.  Individuals that reproduce with K-selected reproductive patterns tend to be better competitors, as they have to survive in dense populations and compete for limited resources.   K-selected populations remain at carrying capacity (K) and grow at a much slower rate.  Individuals with r-selected reproductive patterns are not as competitive, and compensate with high rates of reproduction.  r-Selection most often occurs when conditions are unstable and there are various environmental limitations on a population.  These particular strategists reach K very infrequently, or never (Luckenbill, 1979).

            Research has shown that moisture in the soil is one of the limiting factors for microbes (Tate & Terry, 1980).  Wetter soil essentially is a more favorable environment for microbial communities.  Soils high in moisture allow bacteria to obtain an intracellular solute concentration higher than the solute concentration of the soil, preventing plasmolysis.  Soils low in moisture are less favorable since microbes then have to exert more energy into increasing intracellular concentrations (Stark & Firestone, 1994).  This excessive expenditure of energy reduces the amount of energy exerted toward microbial fitness and competitive ability.     

In this experiment, I observed the reproductive patterns of microbes in wetland and grassland soils.  I hypothesized the microbes from samples of wetland would reproduce with a K-selected reproductive pattern, due to the more favorable, wetter conditions, and the microbes in the drier soil would reproduce with an r-selected reproductive pattern due to constant exposure to less favorable conditions causing these individuals to have a lower competitive ability.  Analysis of reproductive patterns of microbes in the soil gives more insight into conditions that allow for favorable reproductive growth.  This knowledge can in turn lead to a better understanding of the efficiency of soil microbial activity, such as nitrification and break down of organic material.

 

MATERIALS AND METHODS

SOIL SAMPLING

            Point-Quarter sampling technique was used to obtain random samples from both the wetland and grassland in Havenwoods State Forest.  In wetland, the area beneath the dock entrance of the wetland was chosen as the initial starting point for wetland sampling.  The entrance to the grassland area was selected as the initial starting point for grassland sampling.  From that point, while spinning, a Frisbee with an arrow was thrown into the air.  The place where the Frisbee landed was considered the origin of the first quadrant.  A compass (Suunto, model#: A-1000) was used to determine the direction of the quadrant from the initial starting point.  A 30 cm Soil Moisture Meter (model #: 20322) was used to obtain moisture readings of the points where the samples would be collected before every sample.  One sample was taken from each quadrant at a point closest to the origin.  Samples were collected using a 30 cm metal corer.  For every sample, only the soil in the vicinity where the moisture readings were taken were analyzed; the first 20 cm were discarded.   The remainders were stored in sterile120 mL polyurethane cups (No. 37-6015) and labeled.  Once the samples from the first four quadrants were collected, the next starting point was selected by traveling 5 m in the direction of the arrow on the Frisbee.  From that point, the Frisbee was, again, thrown in the air while spinning to determine where the next quadrant would be.  The latter method was repeated twice.  A total of three quadrants were randomly selected.  A compass (Suunto, model#: A-1000) was used to determine the direction of the next quadrant from the previous quadrant.  Since only two samples were taken from the last quadrant, the directions, NE, NW, SE, & SW were placed in a hat and pulled to determine which two quadrants would be sampled.  A total of 10 samples were collected from the grassland, and 10 samples were selected from the wetland.  The Soil Moisture Meter was calibrated before obtaining each reading, and all moisture readings were recorded.  The wetland samples were collected and processed on the first day, and the grassland samples were collected and processed the day after.

 

MEDIA

            Media were prepared and cultivation techniques were performed as described in Janssen et al., 2002 with slight modifications as follows:

 

Dilute Nutrient Broth (DNB):           

0.04 g of Difco dehydrated nutrient broth (BD Diagnostic Systems, Sparks, Md., Cat No. CP146B) was added to 500 mL of distilled water in a 1 L flask (Pyrex, No. 4990) and mixed using a Teflon magnetic bar and hotplate stirrer (Corning, PC-351) until broth was completely dissolved.  50 pop top test tubes (Pyrex No. 9820) were filled with 9mL of DNB and capped with polyurethane closures (Kim Kap, 16 73660).  DNB was sterilized in an autoclave (Hirayama, Model HA-300MI-R) at 121 degrees Celsius for 15 minutes at 5.4 kg of pressure, cooled, and refrigerated until used.

 

Agar plates:

 15 g of dehydrated agar powder (Cat. No. CD 900B) and .066 g of Anhydrous, flaked CaCl2 (B-mesh) was added to 1 L of sterile distilled water in a 2 L flask (Pyrex, No. 4980).  Solution was mixed with a Teflon magnetic bar on a hot plate stirrer (Corning, PC-351) and heated on High until CaCl2 dissolved evenly into solution.  Agar was sterilized in an autoclave at 121 degrees Celsius for 15 minutes at 5.4 kg of pressure.  Agar was poured into 50 pre-sterilized Petri dishes (Kord Valmark) which had been previously placed under UV light for further sterilization.  Once agar solidified, plates were labeled and refrigerated until used.

 

CULTIVATION

            Soil samples were taken to the lab and processed within 3 hours.  All handling of soil samples was conducted in a Laminar Hood Class II Biohazard Cabinet (Labconco, Cat. No. 36213, 04).  Soil Samples were placed in a 37 degrees Celsius incubator for 20 minutes to partially dry out.  Rocks, grass, and sticks were removed and soil was sieved through a brass sieve (10).  1 g of soil was added to 100 mL of sterile distilled water in 150 mL conical flask to obtain dilution of 1:100.  Soil suspensions were dispersed by mixing with a vortex mixer (Vortex-Genie, Cat. No. 12-812) on speed 8 for five minutes.  1 mL of the soil suspensions were then added to 9 mL of DNB to obtain a dilution of 1:1000.  Test tubes were capped with polypropylene closures and mixed with a vortex mixer on speed 8 for 10 seconds.  1 mL of the soil suspensions were, again, added to 9 mL of DNB immediately, to obtain the dilution of 1:10,000.  Suspensions were immediately plated after the last dilution to ensure the microbes would not settle to the bottom of the test tubes. 200 microliters of soil suspensions were added to agar plates.  Inoculants were spread over the agar with a glass triangle spreading rod.  All preparations were labeled by sample number and day, inverted, and incubated at 25 degrees Celsius in the dark.  10 agar plates were not inoculated and incubated as controls for the wetland and grassland, totaling 20 control plates.

COLONY COUNTS

Colonies were observed and counted every three to four days.  Colonies were counted on the same day, resulting in the grassland samples to be counted one day less of incubation than the wetland samples.  Colonies were observed using Darkfield Quebec Colony Counter (Model: 3325) and tallied using Multiple-Tally Denominator (Model: D15557).

ANALYSIS

            A 1-tailed, Independent T-Test was conducted on the results using Excel to compare growth between grassland and wetland cultures every three to four days. Counts of both grassland and wetland cultures were done on the same day.  The number of days of incubation for grassland cultures and wetland cultures were combined into two day intervals for analysis since grassland samples were plated the day after the wetland samples were plated.  The higher number in the interval represented the wetland samples and the lower number in the interval represented the grassland samples (Fig 1).  

RESULTS

            All soil samples were totally saturated, having a Soil Moisture Meter reading of 10.  No colonies were formed on the twenty control plates eliminating the possibility of contamination.  Colonies began to grow after 2 days of incubation.  The average colony counts for the wetland and grassland are represented below (Fig. 1). The colony counts for the wetland on day 5 had an average of 63.1 with a standard deviation of 40.69; Day 8 had an average of 147.4 with a standard deviation of 81.69, and day 12 had an average of 172.4 with a standard deviation of 78.19.  The colony counts for the grassland on day 4 had an average of 203.4 with a standard deviation of 92.53, day 7 had and average of 275.6 with a standard deviation of 115.15, and day 11-12 had an average of 287.4 with a standard deviation of 96.75 (Note: samples 11-15 were counted on day 11 and 16-20 were counted on day 12).  The microbes from the samples collected in the grassland grew significantly faster than the microbes obtained from samples collected in the wetland (Fig 1, p<1.2 x 10-6).  Colony growth on both plates appeared to slow down after the 7-8 day interval.

Fig. 1. Comparison of the number of colonies grown per day between wetland and grassland samples.


DISCUSSION

            The significant difference between the growth of microbes from grassland and microbes in wetland may very well be indicative of environmentally induced growth patterns. In order to confirm this inference, more information needs to be obtained regarding the types of bacterial colonies that were formed.  In this study, there was no deciphering between colonies, which becomes a limitation.  There is no way of indicating whether or not some of the colonies from the grassland samples may have demonstrated k-selected growth patterns or if colonies from the wetland samples demonstrated r-selected growth patterns since colony counting was done under the assumption that all colonies were of the same species.  Klappenblach et al. (2000) observed the time in which bacteria respond to variables in their environment is correlated with the number of rRNA operon genes in their DNA.   More analysis of this aspect will give more insight into whether or not the displayed reproductive patterns in this study were solely environmentally induced or if there may have been some other biological factor involved.  Furthermore, soil moisture may not have been the only limiting factor. Researchers have found that low nutrients in the soil cause microbes to grow slower due to lack of sufficient resources for metabolic activity (Klappenblach et al., 2000).  Soils in the wetland may have been leached of the necessary nutrients, causing the microbes to adapt to growing at a slower rate. In addition, microbes in saturated soils like wetlands tend to be anaerobic (Tate & Terry, 1980).  More techniques can be used to isolate the aerobic and anaerobic microbes to analyze the differences in their reproductive patterns.

            When cultivating the microbes, the wet weight of the soil was obtained rather than the dry weight since the samples were not completely dry.  This may have lessened the amount of microbes in the dilutions which may have been the reason for no visible colonies formed during the 1-2 Day interval.  The dry weight should be obtained to observe whether or not it will result in faster colony formation.  In addition, the colonies were incubated for three weeks.  Literature supports an incubation of 10 weeks for maximum colony growth (Janssen et al., 2002).  Consideration of these methods should be given when replicating this study.

            On both days of collecting samples, it rained.  This is the cause for the total saturation of the soil in both the grassland and wetland.  The amount of rain to each site is variable. Furthermore, because bacterial generations occur very rapidly causing fast adaptations to their environment, the addition of moisture for two days may have altered the reproductive patterns of the microbes in the grassland. Further research should be conducted on days when it has not rained to observe the reproductive patterns under normal soil conditions.

 

References

 

Luckinbill, L.S. (1979). Selection and the rK continuum in experimental populations of protozoa. The American Naturalist, Vol. 113, 427-437. Retrieved on 10/2/07 from Jstor.

 

Tate III, R.L., & Terry, R.E. (1980). Variation in microbial activity in histols and its relationship to soil moisture. Applied and Environmental Microbiology, Vol. 40, No. 2, 313-317. Retrieved on 10/2/07 from http://aem.asm.org

 

Stark, J.M., & Firestone, M.K. (1994). Mechanisms for soil moisture effects on activity of nitrifying bacteria. Applied and Environmental Microbiology, Vol. 61, No. 1, 218-221. Retrieved on 10/2/07 from http://aem.asm.org

 

Klappenblach, J.A., Dunbar, J.M., & Schmidt, T.M. (2000). rRNA operon copy number reflects ecological strategies of bacteria. Applied and Environmental Microbiology, Vol. 66, No. 4, 1328-1333. Retrieved from PubMed.

 

Janssen, P.H., Yates, P.S., Grinton, B.E., Taylor, P.M., & Sait, M. (2002). Improved culturability in pure culture of novel members of the divisions of Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia. Applied and Environmental Microbiology. Vol. 68, No. 5, 2391-2396.  Retrieved on 10/2/07 from http://aem.asm.org