Presence of Microbial Indicators in Reid Park Wetlands

Jepson Sutton

Scott Stine

SWES 574

Purpose

• Determine the effect of water flow rate on microbial indicator concentration– Stagnant Pond, Turbulent Pond, and Waterfall

• Determine the effect of aquatic plants on microbial indicator concentrations– Open water vs. Planted area

• Presence/Effect of chlorination and pH• Compare presence of coliforms and enterococci

Experimental Problems

• It was determined that the ponds contained a shading dye that eliminated all plant growth.

Methods

• Collect samples from three varied areas of constructed wetlands at Reid Park– Located at 22nd Street and Country Club

• Use Colilert and Enterolert (IDEXX) to determine indicator concentrations

Methods

• Make dilutions and add Colilert/Enterolert reagents

Methods

• Pour into Quantitray (IDEXX) and seal using sealer

Methods

• Count number of positive wells

• Enumerate using MPN table

Total Coliforms

• Includes Escherichia, Citrobacter, Enterobacter, and Klebsiella

• Used as a standard for assessing fecal contamination of recreational and drinking water.

• Problems in accuracy due to ability to replicate in environment.

• Colilert positive=Yellow well

Fecal Coliforms

• Includes Escherichia and Klebsiella

• Definitive indication of fecal contamination

• Unable to replicate in the environment

• Colilert can enumerate E. coli concentrations

• Colilert positive=fluoresce under UV lamp

Colilert

Colilert (IDEXX)

• Defined substrate technology (DST) simultaneous detection of total coliforms and E. coli

• Contains specific indicator nutrients: ortho-nitrophenyl-beta-D-galactopyranoside (ONPG) and 4-methyl-umbellifery-beta-D-glucuronide(MUG)

Colilert (IDEXX)

• Total coliforms react with ONPG to form yellow color.

• E. coli react with MUG to form fluorescent color

• Can detect low levels of total coliforms and E. coli (1CFU/100ml)

Total Coliforms

0

50

100

150

200

CFU/ml

Pond 1 Pond 2 Waterfall

Total Coliforms

E. coli

0

5

10

15

20

CFU/ml

Pond 1 Pond 2 Waterfall

E. Coli

Fecal Streptococci Indicators

• Fecal Streptococci have advantages over coliform indicators:– they rarely multiply in water– they are more resistant to environmental stress

and chlorination– they generally persist longer in the environment

Enterococci

• Members of Lancefield Group D of Streptoccocus (fecal streptococci subgroup)

• “Enterococci” - normal colonist of the human large intestine

• Streptococcus often given as genus of this group in water industry

Enterococci

• Includes Enterococcus species faecalis, faecium, avium, and durans

• E. faecalis:– infects elderly patients undergoing surgery

• urinary tract, wounds, blood, appendix, intestinal structures

– emerging nosocomal opportunist due to:• rising incidence of multi-drug-resistant strains

• ease of person to person transfer

Enterococci

• Have been suggested as indicators of:– presence of enteric viruses in environment

• biosolids and seawater

– risk of gastroenteritis for recreational bathers

Enterolert (IDEXX)

Enterolert (IDEXX)

• Uses a nutrient-indicator to detect enterococci– fluoresces when metabolized by enterococci

• Compared to standard membrane filtration (MF) method:– 50% fewer false positives– 95% fewer false negatives

Results

0

0.5

1

1.5

2

2.5

3

3.5

cfu/mL

Pond 1 Pond 2 Waterfall

Enterococci

pH / Chlorine

pH Total Cl (mg/L)Pond 1 8.32 0Pond 2 8.15 0Waterfall 8.24 0

FC/FS Ratio

• >4.0 = contamination of human origin

• <0.7 = contamination of animal origin

• Pond 1: FC/FS = 5

• Pond 2 : FC/FS = 15

• Waterfall : FC/FS = 0.8

Discussion

• Probable pathogen removal factors– temperature, natural die-off, sedimentation

• sedimentation most significant

• Studies have shown slower flow rates allow more settling of microorganisms– Pond 1 would be expected to have lowest

numbers

Discussion

• The highest amount of contamination in all categories occurred in Pond 2– Area where ducks gather

• Total coliforms were present in the largest amounts in all samples– Total coliforms broadest of groups assayed

• Presence of microorganisms could vary over time

Discussion

• Pond 1 and Pond 2 have similar distributions in all categories

• Waterfall does not follow the trend– Total coliforms higher compared to other

samples

• Why?– Turbulence– Cycling of water

Discussion

• FC/FS Ratio suggests:– Pond 1 and Pond 2 contamination is of human

origin

– Waterfall contamination is predominantly of animal origin

Discussion

• Values used to calculate ratio are not completely representative of FC and FS– E. coli is not only member of FC group– Enterococci is only a subgroup of FS

• Ratio only valid for recent (24 hrs) fecal pollution

• Validity of ratio has been questioned