Influence of heavy rainfall on microbial communities and its effects on the San Luis Obispo Creek Watershed

Steven Liu and Matthew FlateboCalifornia Polytechnic State University, Winter 2014

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AbstractMicrobial communities are used to monitor water quality. The presence of microbial communities may also indicate the presence of pathogenic bacteria which may contribute to waterborne illnesses when humans consume or are exposed to contaminated water. During heavy rainfall, there is a marked increase and abundance in microbial diversity. The water movement is responsible for resuspension of the sediment leading to increased bacterial and viral diversity. Furthermore, heavy rainfall leads to urban and agricultural runoff which is a significant contributor of microbes to natural waters. The San Luis Obispo Creek runs through urban and agricultural lands and is an important source of water for local agricultural use and recreational purposes. Water samples were collected from the San Luis Obispo Creek and plated to assess microbial diversity. Our findings demonstrate a two-fold increase in bacterial abundance and diversity after rainfall. While further testing remains to confirm whether fecal coliforms are present in the water, it is still plausible that rainfall is depositing fecal coliforms in the water given the urban and agricultural land surrounding the creek. We recommend careful monitoring of the San Luis Obispo Creek after rainfall and advisories deterring use of the water after heavy rainfall due to elevated microbial contamination.

IntroductionThe Clean Water Act requires coastal states to enforce bacterial water standards that reduce human health risks such as consumption of contaminated water or contact with polluted recreational water (USEPA, 2003). These standards are based on monitoring concentrations of microbial communities. Aquatic microbial communities are typically comprised of fecal coliforms, Escherichia coli, fecal streptococci and enterococci. However, it may also include bacteria of non-fecal origin such as Citrobacter, Klebsiella, various protozoans and parasites. Coliform bacteria exist naturally in the aquatic environment, soil and vegetation. Although fecal microbial communities may not be intrinsically pathogenic, they are abundant in human and animal feces where pathogenic bacteria are found (Noble et al. 2003). Thus high concentrations of microbial communities are strongly linked to an increased abundance of pathogens present in the water. Some of the principal bacterial pathogens that may be present include Legionella, Vibrio, Salmonella and enterohemorrhagic E. coli (Lederberg 2000). Microbial communities are therefore important indicators of waterborne diseases caused by these pathogens. According to the World Health Organization, waterborne diseases account for 4.1% of the global burden of disease and is responsible for approximately 1.8 million human deaths annually. In 2010, the California Water Quality Assessment reported that approximately 29,874 miles of rivers and streams are impaired due to microbial contaminants (USEPA 2010). One of the primary illnesses associated with exposure to pathogenic bacteria in surface water is gastroenteritis; it was reported in 68% of outbreaks related to contaminated drinking water or exposure to recreational water (Dziuban et al. 2006).

Some of the common sources of microbial communities include runoff from urban and agricultural areas, leaking septic systems and sewer lines and sewer overflows. Water pollution should be further exacerbated following a rain event or storm leading to greater concentrations of microbial communities. The sewage transmission infrastructure may be overwhelmed during heavy rainfall leading to leaks in sewers and septic systems which result in transport of

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enteroviruses and enterobacteriae into natural waters (Ferguson et al. 2003). The U.S. Environmental Protection Agency estimates that more than 850 billion gallons of combined sewage are released into the nation’s waterways annually (USEPA 2004). Nonpoint sources such as manure runoff from agricultural areas are also responsible for microbial contamination of water bodies. Rainfall may be an important contributor to the release of pathogens from fecal matter into surface and ground water. As the intensity of rainfall increases, water flow also increases which positively affects the flow of bacterial contaminants through the soil. The rainfall may also disturb the soil leading to the elution of viruses from soil matter. Resuspension of the sediment would result in a subsequent increase of microorganisms in the water (Stevik et al., 1999).

The San Luis Obispo Creek Watershed drains from approximately 84 square miles of land surrounding San Luis Obispo and empties into the Pacific Ocean near Avila Beach. The headwaters of the San Luis Obispo Creek begins from the Santa Lucia Range and is served by eleven tributaries. More importantly, the San Luis Obispo Creek is an important water source for local agriculture. This is significant because contamination of crops due to use of microbially contaminated water may transmit disease to consumers and farm workers. Furthermore, the San Luis Obispo Creek also offers recreational opportunities which may be jeopardized by contamination by increased levels of pathogens as indicated by increased levels of microbial communities (LCSLOC, 2002). For effective public health management, there needs to be an understanding of the influence of meteorological events on microbial communities. This course project intends to investigate the effect of rainfall on microbial communities in the San Luis Obispo Creek. After rainfall or a storm, we expect to observe significantly higher concentrations of microbial communities in the San Luis Obispo Creek.

Materials and MethodsSample site selection. An ideal study site includes urban and agricultural areas in order to accurately represent the effects of manure runoff and storm overflow of the sewer infrastructure due to heavy rainfall. We chose a site in downtown San Luis Obispo near Mission Plaza where the San Luis Obispo Creek flows through. In addition, Stenner Creek (one of San Luis Obispo Creek’s tributaries) flows through agricultural land surrounding California Polytechnic State University and continue through urbanized areas of San Luis Obispo before emptying into the San Luis Obispo Creek downtown. This site should reflect the effects of rainfall on microbial communities due to manure runoff from agricultural lands and storm overflow of sewer lines from urban areas.

Sampling procedures. Samples were collected in sterile 500 mL polypropylene bottles at theSan Luis Obispo Creek downtown near the Mission Plaza. A single sample of water was collected before a precipitation event and well after any rainfall to prevent confounding our results with those of a previous precipitation event. A second sample was obtained following heavy rainfall from the same site. All samples were cooled to prevent the growth of any additional microbes.

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Microbial community assay. The water samples from the creek were plated 72 hours after collection. Bottles containing the samples were mixed by inversion in order to ensure a homogeneous solution. Serial dilutions of the samples were made by preparing 990 µl of LB broth and then adding 10 µl of the water sample. Sequential transfer of 10 µl to the next tube was used to generate dilutions of 10-2, 10-4 and 10-6 with mixing between each transfer. 100 µl of the before and after rainfall samples and 100 µl of each dilution was plated onto trypticase soy agar (TSA) plates and incubated at 37°C for 24 hours. TSA plates were selected because it is a general purpose medium providing the necessary nutrients to allow for a wide variety of microorganisms to grow. On the following day, colony-forming units (CFU) were enumerated on the plates. Colony morphology was also observed and recorded as an indicator of distinct communities of microbial species growing on the plates.

Statistical analysis of results. A Shannon-Weaver diversity index will be used to characterize microbial diversity in the community. It accounts for both abundance and evenness of the species present, providing important information about rarity and commonness of species in a community. A rarefaction curve will also be generated to specifically assess and estimate species richness. Finally, a student’s t-test will be conducted to determine whether microbial communities are significantly different before and after heavy rainfall. All calculations for statistical tests will be generated in R, a free statistical computer program.

Results

The undiluted plates for both before and after rainfall yielded the best number of CFU counts. 300 CFUs were counted on the after rain plate and 55 were counted for the before rain plate. Observation of colony morphology showed ten distinct species for after rain and five distinct species for before rain plate as shown in Fig. 1.

A) B)

Fig. 1: Plates for counting CFUs. A) The before rainfall plate. B) The after rainfall plate. Total abundance in plates increased after rainfall.

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The Shannon-Weaver diversity index, which was conducted to compare diversity before and after rainfall in the stream, gave an index of 1.329218 for before rainfall and 1.746280 for after rainfall. Using the abundance of the different species found on the plates, a rarefaction curve was plotted (Fig. 2). Finally the results of the t-test showed that average number of individuals for each species was not significantly different before rainfall and after rainfall (t-test: t = -1.9772, df = 9.832, p = 0.07672).

Fig. 2: Rarefaction curve for before rainfall and after rainfall, which shows the average species richness plotted against the number of individuals in a subsample.

Discussion

Both undiluted plates of before and after rain showed ideal numbers for CFU enumeration. The after rain plate however showed a much greater abundance of total CFUs, around five times higher. This increase in microbial abundance is most likely attributable to an increase in bacteria abundance in the stream due to rainfall. This is because samples were collected from riffles, so the dirt beneath the water is unable to settle and collect heavier bacterial bodies as opposed to a pool. Because the ground under the water is already being disturbed from the riffles, the increase in abundance must have come from the runoff from rainwater. This alone does not warrant much concern, as increases in microbe abundance are to be expected with rainwater carrying so much debris from neighboring locations.

Because we did not have multiple samples from which to calculate our diversity index, we are unable to quantitatively compare diversity before and after rainfall with any statistical significance. The Shannon-Weaver diversity index does however show an increase in diversity

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after rainfall which is what was predicted. The rarefaction curve plotted shows asymptotic saturation, which because the sampling techniques were carried out uniformly, shows that there was no need to use any sample estimates. Average number of individuals per species was not statistically significantly, so though total abundance was increased after rainfall, there was no difference in their average individual counts for the species found before and after rainfall.

There was a two-fold increase in species found on the after rain plates when compared to the before rain plates. This increase was most likely due to runoff from surrounding areas, whether from the agricultural lands at Cal Poly or from drainage systems in the urban environment that is downtown San Luis Obispo. While not exactly a surprising result, this should raise some concern as such diversity could be due to increased microbial communities because of the locations that the creek water must flow through. It is also conceivable that the increase in diversity is just due to rainwater transporting microbes not commonly found in creek water. Future tests must be done like screening for fecal coliforms after rainfall, to ensure that the bacteria that appear as a result of runoff from rainwater are not a serious contamination.

This experiment showed an increase in diversity and abundance in microbe communities after rainfall. Though these results alone do not show that runoff from rainfall is depositing fecal coliforms from waste systems and agricultural land, they do show that this is a possibility. Given the surrounding land that the creek flows through, future screenings for fecal coliforms are recommended.

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