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Published online 5 July 2005
Published in J Environ Qual 34:1328-1336 (2005)
DOI: 10.2134/jeq2004.0245
© 2005 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
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TECHNICAL REPORTS

Surface Water Quality

Bioluminescent Bacteria as Indicators of Chemical Contamination of Coastal Waters

M. E. Frischer*, J. M. Danforth, T. F. Foy and R. Juraske

Skidaway Institute of Oceanography, 10 Ocean Science Circle, Savannah, GA 31411

* Corresponding author (frischer{at}skio.peachnet.edu)

Received for publication June 27, 2004.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ratio of bioluminescent to total bacteria (bioluminescent ratio, BLR) as an indicator of a variety of types of anthropogenic contamination of estuarine ecosystems was evaluated through a series of laboratory and field studies. Laboratory studies indicated that the BLR of natural bacterioplankton communities was proportionally reduced in the presence of a number of contaminants including diesel fuel and saltmarsh sediments co-contaminated with mercury and polychlorinated biphenyls (PCBs). Bioluminescent ratio inhibition was observed after short-term exposure to a contaminant suggesting a physiological rather than a population response of native microbial communities. Simulated eutrophication did not suppress the BLR. Field observations of the BLR were conducted weekly for a 2-yr period in the Skidaway River estuary, Georgia, USA. These observations revealed considerable seasonal variability associated with the BLR. Bioluminescent ratios were highest during the summer (25 ± 15%), lower in the fall (6 ± 5%) and spring (3 ± 2%), and near zero during the winter. Although the BLR was not significantly correlated to salinity at a single site (Skidaway River estuary), the BLR was significantly correlated with salinity when sites within the same estuary system were compared (r2 = 0.93). Variation in BLR was not correlated to standard bacteriological indicators of water quality including total and fecal coliform bacteria. Comparison of the BLR from impacted and pristine estuarine sites during the fall suggested that anthropogenically impacted sites exhibited lower BLR than predicted from salinity versus BLR relationships developed in pristine systems. These observations suggest that the BLR could be used as a simple and reliable initial indicator of chemical contamination of estuarine systems resulting from human activity.

Abbreviations: BLR, ratio of bioluminescent to total bacteria (bioluminescent ratio) • PCB, polychlorinated biphenyl


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
UNITED STATES CENSUS DATA indicate that approximately half of the U.S. population currently lives in coastal counties and that coastal populations will increase by 15% during the next decade (Culliton et al., 1990; Frey and Speare, 1992). Globally, population densities in coastal regions are estimated to be nearly three times higher than the global average density (Small and Nicholls, 2003). As coastal communities grow, demand for development and infrastructure will continue to climb, further threatening to reduce environmental quality of estuarine systems. However, except for direct measures of identified contaminants, there are few biologically relevant and simple general indicators of coastal water quality available to assess the effect of human activities on the functioning of coastal ecosystems, particularly indicators of chemical contamination (Frischer and Verity, 2005).

To evaluate the condition of estuarine ecosystems, it is logical to focus on indigenous microorganisms for two fundamental reasons. First, the composition and structure of microbial communities are fundamental indicators of ecosystem status. Second, microbial communities are a central ecosystem component integral to the function of all biogeochemical processes. Microorganisms are responsible for the regeneration of nutrients and the transfer of primary production from phytoplankton to microzooplankton and to larger organisms (Sherr and Sherr, 2000).

Several studies have suggested that the ratio of luminescent bacteria to total heterotrophic bacteria (plate counts) might be a useful indicator of anthropogenic impact in estuarine ecosystems (Ramaiah and Chandramohan, 1993; Bacci et al., 1994; Sbrilli et al., 1997; Nocciolini et al., 2000; Perego et al., 2002). Marine luminescent bacteria are a ubiquitous and diverse group of indigenous Gram negative marine heterotrophic bacteria (Hastings and Nealson, 1981; Ramesh et al., 1990). At least eight species in three genera (Photobacterium, Vibrio, and Alteromonas) of marine luminescent bacteria have been reported (Ramesh et al., 1990). The suppression of the luminescence phenotype by a large number of diverse pollutants is a well-known phenomenon and has been exploited in commercial assays to monitor for various organic and inorganic chemical contaminants in a wide variety of environments and sample types (Williams et al., 1986; Steinberg et al., 1995; Doherty, 2001). However, these assays do not use indigenous bacteria and are conducted under controlled laboratory conditions that are not generally representative of in situ conditions. In pristine marine environments, including estuarine and coastal areas, luminescent marine bacteria can account for as much as 50% of the total heterotrophic bacteria that can be grown on standard marine agar media (Hastings and Nealson, 1981). Ramaiah and Chandramohan (1993) reported that in the presence of a wide range of pollutants that result from a variety of different human activities, the number of luminescent bacteria can be greatly depressed. Unfortunately, in this study, the effect of different types of contaminants on luminescent bacteria was not investigated. Determination of the ratio of bioluminescence to total plateable bacteria (BLR) is a potentially attractive indicator system for coastal systems since it targets native microorganisms and is a simple low-cost assay that does not require the use of sophisticated equipment. However, the BLR is influenced by a large number of natural variables that can complicate the interpretation of BLR results as an indicator of ecosystem status. Little is known concerning the natural variability of the BLR in coastal environments.

In this study we report the results of laboratory and controlled field studies designed to elucidate the relationship between the BLR, seasonal variability, and specific contaminants in subtropical estuarine environments.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of Water Samples
Water samples from just below the surface were collected and processed immediately in the field or transported at ambient temperature to the laboratory. All samples were processed within 1 h of collection.

Sampling Sites
To ascertain the variability of the BLR over seasonal time scales, a routine monitoring program was established in the Skidaway River estuary. Water samples were collected approximately weekly within 1 h of high tide from the Skidaway River estuary at the Skidaway Institute of Oceanography's main dock, Savannah, GA (31°35'31.2'' N, 81°00'43.2'' W). Water samples were collected from September 1999 through March 2002.

To estimate the site-specific variability of the BLR, water samples were collected from seven additional estuarine and oceanic sites in the vicinity of Savannah. Samples were collected during a 3-wk period in the fall of 1999 from six locations in Chatham County, GA, and offshore on the South Atlantic Bight continental shelf (Fig. 1) . Samples across the salinity gradient (approximately 3–30 psu [practical salinity units]) of two estuarine rivers in southern Georgia, the Satilla and Altamah rivers, were collected from a small boat in December 1999 and November 2000, respectively.



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  		Fig. 1. Map of sampling locations in Chatham County (Georgia, USA) and the South Atlantic Bight.

 
To assess whether the BLR could be used as an indicator of anthropogenic impact, the BLR was monitored for a 6-mo period at three locations with known histories of human impact. A large marina, Thunderbolt Yacht Sales (formally Palmer Johnson Marina), located at 31°06'43.2'' N, 81°15'36.0'' W, was chosen as a site impacted by human activities. The Thunderbolt Yacht Sales marina is located on the Wilmington River on the intercoastal waterway and is one of the largest marinas in the Savannah area serving both commercial and private clientele. Surface waters are visibly contaminated with hydrocarbon fuels. Analyses of contaminants present in the marina are unavailable, but are likely similar to other well-characterized marina environments (Kiddon et al., 2003; Paul, 2003). Richardson Creek, a tributary to the Wilmington River running past Oatland Island (32°15'36.0'' N, 81°08'52.8'' W), was selected as a reference pristine site. Oatland Island is surrounded by approximately 1700 km2 of undeveloped saltmarsh and supports a small residential community and an environmental education center operated by the Savannah public school district. Richardson Creek was sampled from the dock at the Oatland Island Education Center. The Skidaway River estuary at the Skidaway Institute of Oceanography was chosen as an intermediately impacted site. Recent development of a residential community on Skidaway Island (The Landings) is believed to have contributed to increased nutrient loading of the Skidaway River (Verity, 2002a, 2002b). Samples from Thunderbolt Yacht Sales marina (Wilmington River estuary, Savannah, GA) and Oatland Island Education Center (Richardson Creek, Savannah, GA) were collected approximately weekly from September 1999 through March 2000. Water was collected along the salinity gradient in the Satilla River estuary from St. Catherine's Sound upstream to near the freshwater head (salinity approximately 3 psu) during the late fall of 1999 (6–7 December) and from the Altamah River, GA, in the fall of 2000 (November 8).

The Bioluminescent Ratio (BLR)
The ratio of bioluminescent colony forming units (cfu) to total cfu was determined essentially as described by Ramaiah and Chandramohan (1993). Replicate 1- and 10-mL water samples were filtered onto sterile 47-mm, 0.22-µM Millipore (Billerica, MA) filters, shiny side-down, and incubated at 24 ± 2°C for 24 h on artificial seawater agar media (Frischer et al., 1990) supplemented with peptone (5 g/L) and yeast extract (1 g/L). Total cfu were counted and luminescent colonies were enumerated by counting luminescent colonies in a darkened room. The BLR was calculated as the ratio of total cfu to bioluminescent cfu. Salinity and temperature were routinely measured at the same time using a thermometer and a hand-held refractometer, respectively.

Total and Fecal Coliform Bacteria
Total and fecal coliform concentrations were estimated by standard total coliform (Method 9221B) and fecal coliform (Method 9221E) Most Probable Number (MPN) procedures, respectively (Clesceri et al., 1998). Representative coliform bacteria were confirmed by gas production in brilliant green lactose bile broth (Clesceri et al., 1998). Because coliform bacteria are rapidly inactivated in the presence of saltwater (Hernández-López and Vargas-Albores, 1994; Bordalo, 1994), it was not possible to use membrane filtration techniques when testing estuarine water samples since coliform bacteria were not quantitatively recovered from estuarine water on membrane filters (data not shown). Thus, the slightly more cumbersome fermentation MPN techniques were necessitated in this study.

Effect of Diesel Fuel on the Bioluminescent Ratio
The quantitative effect of hydrocarbon contamination on the BLR was determined in a series of short-term laboratory studies. Five independent experiments with surface water collected from the Satilla and Skidaway River estuaries were conducted over a 2-mo period in the fall of 1999. Exposure concentrations of diesel fuel ranged from 0 to 1.0% by volume. Incubations were conducted in 100-mL volumes in loosely capped 250-mL flasks shaking at ambient temperature. Following 3-h incubations, the BLR was determined as described above.

Effect of Nutrient Fertilization on the Bioluminescent Ratio
The effect of increased nutrient concentrations on the BLR was examined similar to the studies described above except that incubations were conducted in 200-mL volumes in loosely capped 500-mL flasks. Three independent experiments, each using surface water collected from the Skidaway River estuary, were conducted over a 3-mo period in the fall of 2003 (October–December). The effects of fertilization with NO3, urea, glucose, and a mixture of a peptone and yeast extract were examined. Concentrations of NO3 and urea ranged from 0.03 to 30 mM, glucose from 0.1 to 100 mM, and peptone and yeast extract from 0.005 to 0.25 g/L and 0.001 to 0.05 g/L, respectively. Following a 3-h exposure to nutrients, the BLR was determined as described above and compared with BLR values of unamended controls.

Effect of Exposure to Contaminated Sediments on the Bioluminescent Ratio
To assess the effect of exposure to contaminated sediments on the BLR of indigenous bacterial communities from the Skidaway River estuary, the BLR was compared in water samples that had passed through a mesoscale simulated marsh system containing pristine or contaminated sediments. Use of these mesocosms provided the opportunity to explore the effects of exposure to contaminants under realistic conditions not achievable in laboratory scale studies. Two mesocosm cells at the Bioremediation Environmental Research Mesocosm (BERM) facility located adjacent to the Skidaway River estuary at the Skidaway Institute of Oceanography were used for these studies (Lee 1997; Frischer et al., 2000; Sauer 2002). Mesocosms were approximately 3 m in length and 1.5 m in width and depth. Each mesocosm contained approximately 3 m3 of saltmarsh sediments overlying approximately 0.3 m3 of drainage stone. Replicate mesocosms, one each containing contaminated saltmarsh sediments and pristine sediments, were used for this study. Pristine sediment was collected adjacent to Groves Creek, Savannah, GA. Groves creek is a tributary of the Wilmington River located on Skidaway Island, GA. Sediments contaminated with mercury and PCBs (primarily as Arochlor 1268) were collected from the LCP Superfund site in Brunswick, GA (Kannan et al., 1997; Frischer et al., 2000; Wall et al., 2001). Mercury and PCB concentrations in these sediments were on the order of 10 µg/g each (Frischer et al., 2000). The mesocosms used in this study were established in October 1998 and planted with the native saltmarsh grass (Spartina alterniflora Loisel.) such that by the time of study they had equilibrated to resemble a native Southeastern U.S. saltmarsh system with respect to a variety of parameters including the density and growth of saltmarsh grass, porewater nutrient profiles, and the distribution and activity of sulfate reducing bacterial communities (Frischer et al., 2000; Gentzler, 1999; Sauer, 2002). Mesocosms were supplied with water from the Skidaway River estuary. Water was passed through a gravel and sand filtration system, pumped to a common holding tank, and gravity-fed to each mesocosm. Mesocosms were maintained on a 12.75-h simulated tidal cycle. At "high tide" the water level was 30.5 cm above the sediment surface. Water drained via gravity either from the surface or through the sediments into a bottom drain located underneath the sediments. An average of approximately 15% of the volume of one tidal cycle could be collected per tidal cycle from the under-drain suggesting that the residence time of porewater in the mesocosms was on the order of 3 d (Sauer, 2002).

Statistical Analysis
Statistical analyses including ANOVA and regression procedures were done with the SigmaStat Version 3.0 software package (SPSS, 2003).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Response of the Bioluminescent Ratio to Contaminant Exposure
The response of the BLR of indigenous bacterioplankton communities to hydrocarbon (diesel fuel) exposure and to saltmarsh sediments contaminated with PCBs and mercury was examined. Both sets of exposures resulted in a significant depression of the BLR relative to control treatments. The decrease in the BLR in response to the presence of fresh diesel fuel was proportional to diesel fuel concentration, regardless of the initial water sample used, and was best fit with a simple two-parameter hyperbolic function (r2 = 0.98). The BLR was reduced by an average of 84 ± 6% when incubated for 3 h in artificial seawater amended with 1% diesel fuel and 44 ± 6% at concentrations in the parts per thousand range (0.1%; Fig. 2) .



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  		Fig. 2. Effect of diesel fuel on the bioluminescent ratio (BLR) of estuarine bacterial communities. Regression line was best fit to a simple two-parameter model: BLR = a x % diesel fuel/b x % diesel fuel (a = 89.81; b = 0.11).

 
Similarly, the BLR of native Skidaway River estuary bacterioplankton communities was reduced after passage through saltmarsh sediments contaminated with PCBs and mercury relative to treatments that were exposed to pristine saltmarsh sediments contained in a replicate mesocosm with pristine sediments. Over a 6-mo period from October 1999 through August 2000, the BLR values in water samples exposed to contaminated sediments were significantly lower than the BLR in water samples passed through pristine sediments (paired t test, p = 0.014). During this time period the BLR varied seasonally from 0 to 0.71 in the control treatment and 0 to 0.52 in the contaminated treatment (Fig. 3a) . Of the 26 independent BLR estimations taken during this period when bioluminescent colonies were detected, 22 (approximately 85%) of the samples were higher in the uncontaminated versus contaminated treatment and 5 (19%) were less than the BLR from the contaminated treatment (Fig. 3b). Although the absolute magnitude of the BLR was greatest during the summer, the magnitude of the difference in BLR throughout the sampling period did not exhibit a seasonal pattern. These observations demonstrate that exposure of estuarine bacterioplankton communities to two different types of anthropogenically generated contamination, hydrocarbons and sediments co-contaminated with mercury and polychlorinated biphenyls, both resulted in suppression in the BLR of native estuarine bacterial communities.



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  		Fig. 3. Effect of exposure to saltmarsh sediments contaminated with mercury and polychlorinated biphenyls (PCBs) (approximately 10 µg/g each) over one tidal period in saltmarsh mesocosms on the bioluminescent ratio (BLR). (A) Comparison of BLR after exposure to contaminated ({circ}) and uncontaminated (•) sediments. (B) Difference between the BLR of indigenous bacterial communities exposed to pristine sediments and the same communities exposed to contaminated sediments. Relative differences were calculated for paired samples from each sampling date as the difference between the BLR from the clean and contaminated samples divided by the BLR of the clean sample. Positive values indicate that the BLR of communities exposed to contaminated sediments was depressed relative to unexposed communities. One sample (24 Apr. 2000) was excluded from this analysis as an outlier.

 
Response of the Bioluminescent Ratio to Nutrients
The response of the BLR of indigenous bacterioplankton communities to nutrient additions was also examined. In all cases (12 experiments), the BLR was either elevated or unaffected by nutrient amendment. Fertilization with two different nitrogen sources (urea and NO3), glucose, or with a complex nutrient mixture (peptone and yeast extract) did not significantly (p ≥ 0.55) alter the observed BLR from control treatments (Fig. 4a–4c) . These observations suggest that, unlike the response of the BLR to chemical contaminants, eutrophication would not result in depression of the BLR.



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  		Fig. 4. Effect of nutrient addition to the bioluminescent ratio (BLR) ratio of estuarine bacterial communities. Effect of (A) nitrate (•) and urea ({circ}), (B) glucose, and (C) a 5:1 mixture of peptone and yeast extract. Error bars represent one standard deviation of the mean. No difference between any of the treatments was statistically significant: nitrate (p = 0.57); urea (p = 0.55); glucose (p = 0.62); peptone and yeast extract (p = 0.9).

 
Seasonal Variability of the Bioluminescent Ratio
Weekly monitoring of the BLR in the Skidaway River estuary at a single location (Skidaway Institute of Oceanography main dock) demonstrated that the BLR varied significantly and reproducibly over a seasonal cycle (Fig. 5) . Highest BLR levels occurred during the summer (June–August) and lowest, often at undetectable levels, in the winter months (December–February). The BLR values during the spring and fall months were intermediate. Generally, the BLR increased with temperature when water temperatures exceeded 14°C, but temperature and BLR were not strongly correlated (r2 = 0.33; Fig. 6a) . At this single site, the BLR was not significantly correlated with salinity (r2 < 0.0002), but the eight maximum BLR values were observed when water salinity was between 29 and 32 psu (Fig. 6b). There was not an observed relationship between the BLR and total bacteria abundance, chlorophyll a, or nutrient concentrations (data not shown).



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  		Fig. 5. Bioluminescent ratio (BLR) of bacterial communities from the Skidaway River estuary from September 1999 through March 2002. The BLR was determined approximately weekly at high tide.

 


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  		Fig. 6. Correlation of the bioluminescent ratio (BLR) of bacterial communities from the Skidaway River estuary from September 1999 through March 2002 with (A) temperature and (B) salinity.

 
Concurrent with monitoring of the BLR in the Skidaway River estuary, total coliform and fecal coliform abundance was determined. The abundance of total coliform bacteria ranged from 0 to 90 per 100 mL with and average of 9 ± 14 per 100 mL, while the abundance of fecal coliform bacteria ranged from undetectable to 34 per 100 mL with an average of 5 ± 7 over the entire study period (Fig. 7) . There was not a significant correlation between the BLR and the abundance of total coliform bacteria (r2 = 0.005) or fecal coliform bacteria (r2 = 0.08). Similarly, coliform bacteria did not vary predictably with season, temperature, or salinity.



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  		Fig. 7. Concentration of total coliform (•) and fecal coliform ({circ}) bacteria concentration in the Skidaway River estuary from September 1999 through March 2002.

 
Variation of the Bioluminescent Ratio with Salinity
Although there was not a strong correlation between salinity and the BLR over time at a single location in the Skidaway River estuary, the BLR was strongly correlated with salinity when the BLR was examined at other sites in Chatham County, GA, and across the salinity gradient in two southern Georgia estuarine rivers. Since most species of marine luminescent bacteria are obligate marine bacteria, this relationship likely reflects the mixing of fresh and saline water in estuarine systems. During a 1-mo period during the fall of 1999, the BLR was estimated at six sites in Chatham County all within 48 km of the Skidaway Institute of Oceanography. Each site was sampled at least three times except for Fort McAllister (Little Ogechee River), which was only sampled once. Salinity at these sites ranged from 6 psu (Savannah River near downtown Savannah) to 36 psu approximately 64 km offshore on the South Atlantic Bight continental shelf (Table 1). Over this large salinity range the BLR correlated remarkably well with salinity (r2 = 0.93; Fig. 8) . In the Satilla River estuary the BLR was determined throughout the river's salinity range and varied from 0.02 near the freshwater head of the river (salinity 3.14 psu) to 0.22 in St. Catherine's Sound (salinity 30.22 psu). Bioluminescent ratios reached 0.53 where the salinity was 36.5 psu approximately 64 km offshore. The variation of the BLR across the salinity gradient in the Skidaway River estuary was not determined. Similarly, BLRs determined at various salinity levels in the Altamah River that were sampled in a single day (November 2000), varied proportionally with salinity (r2 = 0.95; data not shown).


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  		Table 1. Bioluminescence monitoring sites.

 


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  		Fig. 8. Relationship between the bioluminescent ratio and salinity of bacterial communities at five estuarine sites in Chatham County (Georgia, USA), and from 64 km offshore in the South Atlantic Bight. Savannah River at downtown Savannah (•), Little Ogechee River at Fort McAllister ({blacksquare}), Skidaway River estuary at Skidaway Institute of Oceanography main dock ({blacktriangleup}), Isle of Hope Marina ({blacktriangledown}), Tybee Island south beach ({diamondsuit}), South Atlantic Bight approximately 64 km east of the Savannah Sea Buoy ({circ}). Error bars represent one standard deviation of the mean.

 
Bioluminescent Ratio as an Indicator of Anthropogenic Impact
To assess whether the BLR could be used as an indicator of anthropogenic impact, the BLR was monitored for a 6-mo period at three locations with known histories of human impact and compared with BLR values normalized for salinity from the Satilla River estuary determined during the same time period. A local marina, Thunderbolt Yacht Sales, located on the Wilmington River, was chosen as an impacted site; a tributary to the Wilmington River (Richardson Creek), but one that is surrounded by undeveloped saltmarsh, was chosen as a pristine site. The Skidaway River estuary, sampled at the Skidaway Institute of Oceanography campus, was chosen as a third, intermediately impacted site. During the study period the salinity at the Thunderbolt Yacht Sales marina site varied from 18 to 19 psu and the BLR varied from 0.04 to 0.05 at high tide when samples were collected. The Richardson Creek site (Oatland Island) was slightly fresher with the salinity variation from 14 to 15 psu at high tide. The BLR varied from 0.05 to 0.06 at this site. The BLR from the Skidaway River estuary at the Skidaway Institute of Oceanography was also chosen as an intermediately impacted site. Recent development of the island as a golf course community (The Landings) beginning in the late 1970s has likely contributed to increased anthropogenic inputs into the Skidaway River, particularly dissolved organic nitrogen and other nutrients associated with cultural eutrophication (Verity, 2002a, 2002b). Salinity during the fall 1999 period varied from 24 to 26 psu and the BLR varied from 0.07 to 0.10. Comparison of BLR values between the Satilla River, Thunderbolt Yacht Marina, Oatland Island, and the Skidaway River estuary made during the same time period (fall 1999) suggest that the BLR values of both the Thunderbolt Yacht Sales marina and the Skidaway River sites were depressed relative to predicted BLR values based on the Satilla River estuary (p = 0.029 and p < 0.001, respectively). Conversely, the BLR measured in Richardson Creek was not significantly different (p = 0.095) from predictions based on the salinity vs. BLR relationship predicted from the Satilla River estuary (Fig. 9) . These observations support the laboratory studies described in this report that suggest a relationship between the presence of anthropogenically derived contamination and depressed bacterial BLRs.



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  		Fig. 9. Bioluminescent ratio (BLR) of bacteria communities from three estuarine sites in Chatham County (Georgia, USA): Richardson Creek at Oatland Island (•), Wilmington River at Thunderbolt Yacht Sales marina ({blacksquare}), and the Skidaway River at the Skidaway Institute of Oceanography main dock ({blacktriangleup}) were compared with the regression predicted bioluminescent ratio of bacterial communities from the Satilla River estuary ({circ}). BLRSATILLA = (salinity x 0.0125) – 1.21; r2 = 0.73. The bioluminescent ratio from each of the sites in Chatham County was measured from September 1999–March 2000. The bioluminescent ratio of bacterial communities was determined from 6–7 December in the Satilla River. Error bars represent one standard deviation of the mean.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The objectives of this study were to evaluate whether the BLR of indigenous estuarine bacteria populations could serve as a simple, reliable, and general indicator of anthropogenic impact in subtropical estuarine environments. Specifically, in this study we investigated whether the BLR of indigenous estuarine bacteria populations was predictably suppressed by a variety of contaminants associated with human activity and the seasonal variability of subtropical estuarine bioluminescent bacteria populations, and compared the BLR of estuarine populations in a variety of pristine and impacted environments.

Luminescent-based assays that use specific cultures of bioluminescent bacteria (typically V. fischeri) as indicator species are routinely used to detect the presence and toxicity of chemical contaminants in coastal environments (Perego et al., 2002; Doherty, 2001; Long et al., 1998; Steinberg et al., 1995). In this report we demonstrated that mixed populations of indigenous luminescent estuarine bacterial communities could be used as an indicator of some types of estuarine contamination commonly associated with human activity. Similar to culture-based assays such as the Microtox system (Strategic Diagnostics, Newark, DE), these studies also indicate that the BLR is rapidly suppressed by a variety of contaminant types and exposure sources and that the decrease in the BLR is proportional to the concentration of the contaminant. This observation confirms that the contaminant, rather than experimental conditions, was responsible for the observed decrease in the BLR. Since the suppression of the BLR occurred after 3 h of exposure to the contaminant, it is unlikely that the decrease in the BLR reflects a change in the composition of the bacterial community, but rather in the physiological activity of the community and the phenotypic expression of bioluminescence by bacteria capable of bioluminescence. Passage of water through weathered sediments contaminated with mercury and PCBs also suppressed bioluminescence, further demonstrating that the BLR of subtropical estuarine microbial communities responds to a variety of contaminants and contaminant exposure sources. These controlled experiments indicate that the expression of bioluminescence by native estuarine bacterial populations is sensitive to a wide range of contaminant insults that, in the environment, are generally associated with human activities, population growth, and coastal development. Interestingly, increased nutrients did not appear to affect the BLR. Since bioluminescent bacteria tend to be copiotrophic strains that typically thrive under rich nutrient conditions, it seems likely that they were either unaffected or actually stimulated by the simulated eutrophication treatments. These results suggest that the BLR assay is not useful for identifying eutrophication processes.

Several studies have reported on the potential usefulness of monitoring the fraction of bioluminescent bacteria as a sentinel of water quality (Nocciolini et al., 2000; Sbrilli et al., 1997; Bacci et al., 1994; Ramaiah and Chandramohan, 1993). However, to our knowledge, none of these studies have investigated the natural variability in this parameter independent of other factors or systematically examined the response of BLR of native estuarine bacterial communities under controlled laboratory conditions. Despite the quantitative response of the BLR in controlled laboratory experiments, the BLR of natural populations varied considerably over time and space in the absence of any specific contaminant dosing. Thus, if the BLR is to be used as an indicator system, the natural variability of the BLR must be considered and accounted for. In these studies salinity and temperature apparently explained the largest fraction of the variability in the BLR and when these variables were accounted for, the BLR index could be used to differentiate sites known to be affected by human activity. Since bioluminescence is considered to be a property of a relatively small group of obligate marine proteobacteria (Ramesh et al., 1990; Hastings and Nealson, 1981), the fluctuation of BLR with normal environmental variation likely reflects seasonal differences in the composition of the bacterial communities.

Although specific bacterial species, particularly the enteric bacteria, have historically been used as indicators of human contamination of potable water and receiving waters (Frischer and Verity, 2005), recent literature on the subject suggests that these organisms are not necessarily reliable indices of environmental deterioration, contaminant loading, shellfish quality, and public safety of water (Kantor and Rhodes, 1994; but see Vernberg et al., 1992, 1996). Furthermore, it remains unclear whether the presence of human pathogens or pathogen surrogates reflect in any manner the "health" of marine ecosystems (Bacci et al., 1994). Thus, there remains a need to evaluate other microbial parameters as indicators of human activity on aquatic resources, including coastal environments. The results of these studies suggest that the ratio of luminescent bacteria to the total number of cultivatable bacteria in subtropical estuarine environments could be used to detect the presence of contaminants that are independent of sewage contamination and is therefore a useful addition to the suite of indicator systems currently available to assess estuarine water quality and ecosystem health. However, since there can be considerable variability in the BLR associated with other natural sources, particularly temperature and salinity, long-term seasonal baseline data sets will have to be developed to effectively use the BLR criteria as an indicator system.


   ACKNOWLEDGMENTS
 
This work was partially supported by the U.S. National Science Foundation (OPP-00-83381 and OCE 99-82133), and the U.S. Department of Energy (FG02-88ER62531 and FG02-98ER62531). We also wish to acknowledge the work of several students, technical staff, and high school teachers who contributed to these studies including: Victoria Baylor, Susan Ebanks, Deborah Goldberg, Sandra Pagen, and Tina Walters. We also wish to thank Dr. Clark Alexander and Michael Robinson for providing GIS assistance. We gratefully acknowledge the cooperation of the administration and staff of the Oatland Island Education Center (www.oatlandisland.org).


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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