Samples from the study sites from the Rio Grande were all collected on April 6th, 2012 with a temperature of 26.3°C, typical of the Spring season in the South Texas area. The Rio Grande exhibited a flow of 43.0 m3/s (IBWC, 2012) which is the average flow for April but is dependent on the releases into the Amistad Reservoir, located upstream, and the Falcon Reservoir, located downstream, of the study areas (Torres et al., 2012). The year to date precipitation was 3.23 cm as of the month of April. Three study sites were selected to assess if any man-made influences such as effluent discharges (from WWTP), urban, and agricultural runoffs have a direct influence on the water quality and microbial community. Physiochemical factors may result in changes to the bacterial composition affecting the overall quality of the water (Abasiofiok et al., 2011;
Mendoza et al., 2004; Poitelon et al., 2010; Winter et al., 2007).
Three sampling Sites 1-3 (Figures 2.1 to 2.3) were examined along the Rio Grande within the Laredo, Texas area. Total sample site stream length was 8,314 km. The riparian area contained dirt roads, mesquite trees, tall grasses, and vegetation typically found in South Texas floodplains;
near the river’s edge Arundo donax (Carrizo) was also present. Potential anthropogenic influences present were noted (Torres et al., 2012).
Site 1-Zacate Creek Area (Site 1-ZCA) was located downstream of the Zacate Waste Water Treatment Plant (ZWWTP) (27°29’50”.16 N 99° 29’.10”W) at an elevation of 129 m. It is the oldest waste water treatment plant located on the banks of the Rio Grande in the City of Laredo, Webb County, Texas 78040. This plant has an average effluent discharge capacity, during a two hour peak period, of 20,150 gallons per minute (gpm) equivalent to 76,226 liters per minute of treated effluent containing chlorine levels that range between 3.2- 3.9 mg/l with the highest acceptable level at 4.0 mg/L.
Site 2-Bridge I (Site 2-B I) rock outcrop was located upstream of the COMAPA
(Comision Municipal de Agua Potable y Alcantarillado) in Nuevo Laredo, Tamaulipas, Mexico (27° 29’56’.71 N 99°30’ 34.79” W) at an elevation of 110 m. The rock outcrop is an area of visible rocks only when the Rio Grande registers low levels of water flow such as in drought seasons. There are a number of sewage outfalls present between Site 2-BI and Site 1-ZCA (David Negrete, Civil Eng. per. comm.).
Site 3-Jefferson Water Treatment Plant (Site 3-JWTP) intake was located on the concrete platform (27° 31’ 26.31’’N 99° 31’ 27.57” W); the elevation for this site was 118 m. It is the first and the oldest Drinking Water Treatment Plant (DWP) in Laredo, Texas. The Jefferson Drinking Water Plant (DWP) has no known outfalls or effluent sources within 8.45 km upstream of this site. It is the primary drinking water source for the Laredo, TX municipality.
Table 2.1 GPS locations of raw sewage outfalls along the Rio Grande study area (IBWC, 2011).
Outfall discharge(s) indicated with its relative position (upstream/downstream from Site
indicated) and distance, to each of the three individual collection Sites (TAMIU C.E.E.S., 2013).
Outfall LATITUDE LONGITUDE Site 1-ZCA Site 2-BI Site3-JWTP
Figure 2.1 The three collection sites and the nearest Nuevo Laredo, Tamaulipas, Mexico outfalls within the study area. Outfall locations are approximate due to the close proximity of these outfalls. Image was produced using ArcGis Version 9.2 (ESRI, 2006) provided by TAMIU C.E.E.S. (2013).
Sample collections
Samples from each site were collected by grab sampling using a collection pole that would hold
Figure 2.2 Estimated location of study sites superimposed on the map of the study areas for the International Boundary and Water Commission U.S. Section, Texas Clean Rivers Program.
“Bacterial Characterization on the Rio Grande 2304, Laredo, TX and Nuevo Laredo, Tamps, Mexico” (IBWC, 2011). Permitted U.S. wastewater discharges into the Rio Grande-Rio Bravo with the three collection sites are indicated.
Sample bottles of different sizes, as samples were collected they were immediately stored on ice.
All samples were collected from the surface of the river flow at a depth of approximately 50 cm with collection bottles facing towards the river current (downstream). For all three sites, the same
Figure 2.3 Collection Sites 1, 2, and 3 as indicated using GPS coordinates.
Scale not provided (Google Earth, 2013).
Figure 2.4 The twelve sewage outfalls from Nuevo Laredo Tamps, Mexico located within the study area of Sites 1, 2, and 3. Scale not provided (Google Earth, 2013).
quantity and sizes of collection bottles were used: 120 ml Idexx sterile bottles for bacterial counts, 500 ml Nalgene (amber) sterile bottles for nutrient determinations, and 120 ml Idexx
bottles for DNA extractions. All the sample bottles contained the following information:
Collection site, date, time of collection, and required tests. In situ water quality parameters were assessed using a YSI Model 6600multiparameter probe (YSI Incorporated, Yellow Springs, Ohio) previously calibrated according to the instruction manual. The following parameters were taken for each of the three sites: depth, pH, water temperature, chlorophyll a, specific
conductivity, and optical dissolved oxygen (DO).
At Site1-ZCA, and Site 2-B I, the water samples were obtained by walking inward 10-15 meters from the edge of the river bank. Samples were taken when the water level was at least 60-70 cm. When walking into the river, care was taken to not disturb the sediment on either site.
For Site 3-JWTP, this was the only sample collected by standing on the concrete platform and extending the collection pole. The depth of collection was not more than 50 cm in depth.
Total nitrogen
Total Nitrogen was determined for all samples using the method by Bachmann and Canfield (1996). The analysis was done within 28 days of collection. Prior to analysis, samples were acidified with a 0.2% H₂SO₄ to a pH of <2.0. This is a modified method that determines the total nitrogen present within each sample and not the partial sources of nitrogen that may be present. All nitrogen sources, ammonium-nitrogen (NH4⁺), NO2⁻, and or organic nitrogen (Org-N), are oxidized to NO3--N using potassium persulfate (K2S2O8). The Kjeldahl-Nitrogen procedure compares to the E.P.A. (1979) approved method involving the sum of nitrate-nitrogen and Kjeldahl-nitrate-nitrogen which is the Total-N concentration as the sum of the NO⁻3N and the Kj-N concentration. This allows for an estimation of the concentrations of nitrogen as nitrate-nitrogen (NO⁻3-N) plus the measurement of nitrogen found in ammonium–nitrogen, nitrite, nitrogen and organic nitrogen (NH4⁺, NO2⁻, NO-3⁻ and Org-N). The purpose of this conversion is to reduce absorption due to organics within the water. The total nitrogen is calculated using the second derivative of the absorbance at 224 nm.
Each sample was run in triplicate and an average was taken of the readings. To
determine the total nitrogen, a 10 ml subsample was placed into a sterile (25m x 200mm) screw cap tubes; one blank of deionized water was included per set. This was followed by pipetting 1.5ml of oxidizing reagent which contained 6.0g K2S2O8 dissolved in 1.5ml NaOH. Screw caps were placed on all sample tubes and inverted gently 10 times to mix. Tubes were then
autoclaved at 15 psi at 121°C for 30 minutes for complete digestion from nitrogen to nitrate and
allowed to cool at room temperature. Samples were then acidified to a pH of < 2.0 with 0.2 ml of concentrated H2SO4 (98% reagent grade). The pH level for each sample was measured using a VWR 600 pH meter that was calibrated with 3 standard buffers: 4.0, 7.0, and 10.0; the slope was 97.6%, prior to adding the acid (as a preservative). One ml of each sample was pipetted into a 2-cm cell path length cuvette and measured for absorbance with the first reading at 275nm followed by a second reading at 225nm. The spectrophotometer used for these reading was Spectro 23 RS (Loomed, Inc. CA). Standards were used to calculate the OD at 225nm and OD 275nm, with corrections using blanks (dH2O) and the total nitrogen was calculated based on the method by Bachmann and Canfield (1996).
Total phosphorous
The total phosphorous was determined for all samples using the method by Prepas and Rigler (1982). The analysis was completed within 28 days of collection. This method allows for the available phosphorous fraction to be assessed independent from the suspended inorganic phosphorous that might be present in sediment within the water column. Unfiltered water samples were used.
Each sample was run in triplicate and an average was taken of the readings. For each 50 ml sample, 0.4 g of K2S2O8 (potassium per sulfate) was added followed by 0.5 ml H₂SO4 for the digestion reaction. After 45 minutes, the samples were neutralized with 1.5 ml of 10N NaOH.
This was followed by adding 5 ml of reagent solution (stock solution plus reagent solution). The day of the analysis 5.17 g ascorbic acid were added to 100 ml of deionized H2O, followed by 400 ml stock solution. The stock solution, stored in an amber glass bottle, contained the following:
15 g of (NH4)6Mo7O24·4H2O (ammonium molybdate), 0.34 g of K22Sb2 (C4H22O6)2 (potassium antimony tartrate), 140 ml H2SO4 of (sulfuric acid), and 1,650 ml of deionized water. Once the neutralization was attained the absorbance was read at 885 nm within 30 minutes.
Heterotrophic plate counts
For each of the three sites, Site 1-ZCA, Site 2-B1, and Site 3-JWTP intake heterotrophic plate counts (HPC) were performed using R2A agar media (Becton-Dickinson and Co.). Each sample was serially diluted in 9 ml of sterile 0.85% NaCl up to 10-8 and plated in triplicate for a final plate dilution of 10-1 to10-9. An aliquot of 100µl of the raw water sample from each site was used for the 10-1 dilution plate. This served as a positive control for each site and provided an overall idea of the bacterial colony counts. Plates were incubated at room temperature (23°C) in
a HACH incubator. The plates were checked for bacterial growth at 24hrs, 48hrs, 72hrs, and at 96 hours for the final count. Plates chosen for colony counts were those that exhibited an even distribution of colonies. Although, a traditional culture based approach does not facilitate the recovery of the whole bacterial community, the bacterial counts allowed us to assess, in part, the bacterial abundance for each of the selected three sites (Calabria de Araujo and Schneider, 2008 Holben et al., 2004; Ibekwe and Lyon, 2007; Lyautey et al., 2005).
Statistical analysis of total nutrients and heterotrophic plate counts
One-way analysis (ANOVA) was performed on the total nitrogen, total phosphorous, and heterotrophic plate counts using SPSS Version 16.0. When a significant difference (p < 0.05) was detected between sites for each factor, Tukey’s HSD post-hoc test was performed using a significance level of p < 0.05).
DNA extraction of water samples for DGGE analysis
For DNA extraction, samples were collected directly into 50-ml sterile conical tubes, as previously mentioned (see Sample Collections). The samples were kept frozen at -25°C from 4/6/12 until 5/31/12 when samples were processed for DNA extraction. Duplicate 50-ml conical tubes for each site were thawed out at in deionized H2O at ambient temperature. Once samples thawed, all samples were centrifuged for 30 minutes at 9,500 rpm (Beckman Coulter Allegra X-22R) at 25°C to concentrate the bacterial DNA. The supernatant was decanted and 1ml of sterile molecular H2O was added to resuspend the sediment. Samples were vortexed to dislodge the sediment and cells. Centrifugation for 5 minutes was repeated followed by suspension in 1 ml of sterile molecular H2O. The Fast DNA Spin Kit for Soil (Qbiogene, Inc, Carlsbad, CA) was used for the extractions with some modifications for water samples. All disposable materials (tubes, etc.) were autoclaved in addition to being crosslinked. Reagents from the kit were also UV-crosslinked except for the MT-buffer. For each sample, 500 µ1 of the slurry was added to the matrix tube and the procedure followed the manufacturer’s protocol (Qbiogene, Inc, Carlsbad, CA). For the final DNA elution, the filter with 50 µl of sterile molecular H2
O was allowed to incubate overnight followed by vortexing for 30 seconds and
centrifugation for 1 minute at 14,000 rpm. The extracted DNA was transferred to a new PCR tube and frozen at -80°C. A negative control (molecular H2O) was used in the protocol as well.
Quantification of DNA
For each DNA extraction, DNA was quantified using the manufacturer’s protocol for the QuantiFlour dsDNA System (Promega, Madison, WI). All disposable materials (conical tube, microcentrifuge tube, PCR tubes, aerosol barrier tips, etc.) and molecular H2O were UV-crosslinked. The dye was thawed on ice and kept in the dark. The TE (Ethylenediamine Tetraacetic Acid-buffer solution) working solution (1X TE), preparation of the unknown DNA samples, and the preparation of the Quantiflour dsDNA dye working solution (1X) were
prepared according to the manufacturer’s protocols with some modifications. All DNA samples were diluted 40-fold by adding 2.5 µl of extracted DNA into 97.5 µl of the 1X TE. All tubes were wrapped completely with foil prior to adding100µl of the 1X working dye solution. The tubes were left to incubate for 10 minutes immediately following the first addition of dye. After the 10 minute incubation, each PCR tube containing the unknown DNA was placed into the PCR tube adapter (Promega, Madison, WI) and the DNA was quantified at 460 nm using a calibrated curve for the blue fluorescent channel (Turner Biosystems Modulus Single Tube Multimode Reader).
Concentrations of DNA were measured in ng /ml and multiplied by the dilution factor of 80 to convert to ng/µl. Prior to PCR amplification, DNA templates were diluted using molecular H₂O to obtain a final concentration of 0.04 ng/µl for DGGE-PCR.
16S rRNA gene PCR for denaturing gradient gel electrophoresis
Genomic DNA was extracted from the water samples and used for the amplification of the 16S rRNA gene. A 336-bp fragment of the bacterial 16S rRNA gene including the
hypervariable regionV9 of the domain bacteria was amplified from the DNA extracts using the primers 1070F (1055-1070): 5’ ATG GCT GCT GCT AGC T 3’ and 1406R-GC (1392-1406): 5’
CGC CCGCCG CCCGC CCC GCG CCC GGC CCG CCC CCG CCC CAC GGG CGG TGT GTA C 3’with GC clamp (Legatzki et al., 2011). The reaction mix for PCR reactions contained, per 50 µl reaction mix: 23.25 µl of molecular H₂O, 5µl of Qiagen Hot Star Buffer, 5 µl of 5 µM of primers, 5.0 µl of BSA, 4.0 µl of 10 mM dNTPs, 2.5 µL of DMSO, 0.25 µl Hot Star Taq, and 5.0 µl of template (0.4 ng/µl). The final concentrations per 50 µl reaction were: 1X Hot Star Buffer, 0.5 µM of each primer, 400ng/µl of BSA, 200 µM of each dNTP, 5% DMSO, 0.02 U/µl of Hot Star Taq DNA Polymerse (Qiagen, Inc., Valencia, CA, USA), and 0.04 ng/µl of DNA template. The reaction conditions were: 95°C for 15 min, 30 cycles of 94°C for 45 s, 55°C for
45 s, and 72°C for 30 s, followed by an extension at 72°C for 7 min and 25°C for 5 min. The bacterial DGGE ladder was composed of 10 bands generated by 16S rRNA gene fragments (1070F-1406R-GC) from mixed river isolates (C2-4 and C2-6) and two Pseudomonas aeruginosa strains Boston 41501 and ATCC 2785. Bands were visualized and confirmed by running the PCR products on a 2% agarose gel prior to DGGE.
DGGE procedure
All 16S rRNA-DGGE PCR products from the three sites, Site 1-Zacate Creek Area (Site 1-ZCA), Site 2-Bridge I (Site 2-BI rock outcrop), and Site 3-Jefferson Water Treatment Plant (Site 3-JWTP), were run in duplicate. The D Code Universal Mutation System (Bio-Rad Laboratories, Hercules, CA) is a vertical electrophoresis instrument used to run any vertical gel-based mutation detection method. The 100% acrylamide gel contained 7 M of urea and 40% of deionized formamide. The denaturing solutions were prepared with urea-formamide using 45%
for the low and 70% for the high denaturing solutions, according to the manufacturer’s
instructions. The DGGE gel lanes were loaded using 40µl of PCR product. The bacterial DGGE ladders were used as external references at the beginning and at the end of the gel. The ladders consisted of PCR products from mixed river isolates (C2-4 and C2-6) and two Ps. aeruginosa strain Boston 41501 (ATCC 27853). The bands were analyzed and evaluated using Quantity One 4.6.9 (Bio-Rad Laboratories, Hercules, CA).
DGGE band excision
The gel was carefully removed from the DGGE chamber and placed in the SBRY green to stain for 40 minutes and a quick destain in dH2O. After the gel was destained, it was placed on
transparency paper on the Transilluminator U.V. (Bio-Rad Laboratories, Hercules, CA) to excise the selected bands (containing mixed OTUs) that were common to the three sites and those that were unique to each of the three sites. The procedure to excise band from the gel was as
followed: 1.7 ml tubes were cross-linked for 30 minutes followed by the addition of 100µl of the previously prepared elution buffer and cross-linked. The bands were cut out using a sterile scalpel that was flamed with alcohol intermittently between excisions. When all bands were excised, they were transferred to the tubes containing the 100 µl elution buffer followed by incubating the tubes at 50°C for 30 minutes. The elution buffer contained the following: 10 ml of 5 M NH4OAc, (ammonium acetate), 200µl of 0.5 M EDTA, add 85 ml of M.W., pH adjusted to 8.0 with 10 N NaOH. The elution buffer final concentration used was 0.5 M NH4OAc, 1 mM
of EDTA at pH 8.0 (Legatzki et al., 2011). After incubation residual acrylamide was pelleted by centrifugation at 13,000 rpm for 1 minute following the Qiagen protocol. The supernatant containing the DNA for each excised bands was transferred to 0.2 ml PCR tubes that were previously sterilized and U.V. cross-linked. There were a total of 30 bands (now referred to as operational taxonomic units, OTUs) excised. The first 24 OTUs were from Site 1-ZCA. For Site 2-B I and Site 3-JWTP, three unique OTUs were excised from each, separately, for a total of six additional excised bands. The OTUs of these two sites, Site 2 and Site 3, were considered unique OTUs. The supernatant was frozen at -20°C. In this study, the OTUs were not cloned and sequenced for identification but will be in a future study.
DGGE operational taxonomic units (otus) analysis report
The DNA bands obtained from the DGGE gel were evaluated in the following manner. It was the hypervariable region V9 of the domain bacteria which was amplified to target the
microbial community of the selected sites from the Rio Grande- Rio Bravo (Rosario et al., 2007).
After each set (matching bands across the lanes) on the gel from each study site on the image were identified, the OTUs were analyzed using a set of options provided by the software program. These commands allow for the analysis of the bands first by defining the lanes, followed by band detection, band attributes, and band matching. The reports were: Band type report, Similarity Report by Lane and Similarity Matrix Report which both use the Dice Coefficient formula. The hierarchal clustering analysis was completed using UPGMA which produces a phenetic dendogram. Several applications were provided depending on the type of analysis chosen (Quantity One Manual 4.6.9 by Bio-Rad, USA). Under the Comparison Options from Quantity One, all OTUs known and unknown were selected. For this selection there are other options to follow: if comparing similarities in all OTUs, one can select both band position and intensity of the bands, or only band position when comparing samples (Bio-Rad, U.S.A).
OTUs are expressed as an Index that ranges from 0-1(1 suggests indistinguishable bands). The difference lies in the weight of the number of positive and negative matches based on the
distance or similarity that is calculated; for this analysis similarities were calculated on all OUTs (matched, matched unknowns, and unclassified) by lane position only (Bio-Rad, U.S.A.).
For the Dice coefficient analysis, the mathematical equation uses a vector representing the bands found in each lane, vector S contains B elements (S= (s1, s2, and s3….s B) where B represents the number of band types in the lane’s band set: Si is 1 if band if found on the lane; Si
is 0 if the band type is not found. If S and T are two vectors representing two samples that are related isolates then the similarity can be calculated. The Dice Coefficient has been employed in the analysis of similarities of individuals (binary patterns) in the absence of knowledge of
ancestry of all the individuals in the population (Kosman and Leonard, 2005). The Dice Coefficient is also called the coincidence index, since it measures the chance of one species (band) occurring in the same sample (lane) as another, given the second band is present;
basically is the sum of all the bands that appear in both lanes, divided by the sum of all the bands in both lanes and multiplied by 200. In other words, the equation can be explained as the sum of bands that appear in both lanes divided by the sum of all bands in both lanes, multiplied by 200, the question mark (?) represents the sum of all bands. Similarity = 200 x (? (Si, Ti))/ (? (Si + Ti), while the equation for distance is Distance = 100-similarity (Boxall, 1999). The Dice coefficient analysis (Table 3.2) from Quantity One 4.6.9 allowed the similarities/distances to be calculated
basically is the sum of all the bands that appear in both lanes, divided by the sum of all the bands in both lanes and multiplied by 200. In other words, the equation can be explained as the sum of bands that appear in both lanes divided by the sum of all bands in both lanes, multiplied by 200, the question mark (?) represents the sum of all bands. Similarity = 200 x (? (Si, Ti))/ (? (Si + Ti), while the equation for distance is Distance = 100-similarity (Boxall, 1999). The Dice coefficient analysis (Table 3.2) from Quantity One 4.6.9 allowed the similarities/distances to be calculated