Impact of Antibiotics on Denitrifying Biofilm Bacteria
Jeremiah Rocha, Julie Swierenga, Professor David Wunder, Calvin College, Summer 2013
Objective
To understand the impact of low concentrations of
antibiotics on the denitrifying ability of biofilm used
in water filtration through assessment of
denitrification rates and bacterial viability.
250
S inf,5
feed concentra tion (S inf)
S inf,4
200
150
S inf,2
100
S3
S2
S1
SF inf,0
S5
S4
S inf,1
75
50
S inf,3
effl uent concentra tion (S)
Live/Dead Stain
Bacterial Viability
• Using Invitrogen’s Live/Dead Bacterial Viability Kit
L7012, at the end of each run the biofilm was
treated with a fluorescent dye and analyzed using
the Cary Eclipse Fluorometer.
• Based on Live/Dead results, low concentrations
of antibiotics does not have a significant effect
on the viability of the biofilm bacteria.
• Biomass in the reactor after the nitrate sweep
showed an increase at both the low and high
antibiotic concentrations.
• The ratio of green to red fluorescence
represented the ratio of live to dead bacteria in
the biofilm.
5h
r
4h
r
3h
r
2h
r
1h
r
7 da ys
ni tra te s weep
Figure 2. Illustration of the nitrate sweep step-feed conditions. The feed
concentrations of nitrate were increased at 1 hour intervals, and the effluent
concentration was measured after each step to determine nitrate utilization by
the biofilm bacteria.
Experimental Set-Up
• The control run contained no antibiotics in the
influent,
• The “low” antibiotic concentration run contained
3.44 µg antibiotics in the influent.
• The “high” antibiotic concentration run
contained 34.4 µg antibiotics in the influent.
• Antibiotics used: Sulfamethoxazole (SMX) and
Ciprofloxacin (CIP).
Ion Chromatography
• Effluent water was collected daily.
• Samples of the effluent were collected before
and after each nitrate sweep.
• All samples were analyzed for nitrate via Ion
Chromatography.
Live:Dead
Ratio
Figure 5. 96-Well Plate with
biofilm samples dyed with the
Live/Dead stain.
Results
Denitrification Rates
70.8
0.43
Run 2
3.44
138.0
0.41
Run 3
34.4
2640.7
0.41
Utilization Rate (mg/L)
6
5
4
3
1
Volatile Solids Test
0
Acknowledgments
Control (No Antibiotics)
3.44 ug/L (SMX and CIP)
2
Viability Analysis
• Low concentrations of antibiotics (3.44
µg/L and 34.4 µg/L) decreased the
utilization of nitrate in a denitrifying biofilm
by approximately 50% when present in
water.
• Biomass in the reactor showed an increase
with the introduction of low and high µg/L
concentrations of antibiotics.
• Future community structure analysis will
provide complementary data.
Mr. Bob De Kraker, Calvin College
Mr. Rich Huisman, Calvin College
Mr. Phil Jasperse, Calvin College
Ms. Lori Keen, Calvin College
Mr. Scott Prentice, Calvin College
Wyoming Wastewater Treatment Plant
7
• Biofilm was harvested from the CFRAB and
combusted in an oven to determine volatile
solids, which represent biomass.
0
Conclusions
Nitrate Utilization Curves
Figure 3. Cartridges for the IC. Effluent samples were filtered and
treated with Benzalkonium Chloride (BAC) before being run through
the IC.
Run 1
Figure 4. Aluminum trays after
combustion in volatile solids analysis.
• Denitrification rates in a typical untreated
drinking water NO3 concentration range (50
mg/L to 70 mg/L) was decreased by
approximately 50% with both low and high
concentration antibiotics conditions.
• Continuous-Feed Rotating Annular Bioreactors
(CFRABs) were run for 7 days to simulate
biofiltration of drinking water.
• Mineral water, acetate, nitrate, and antibiotics
were fed through the CFRAB which was seeded
with biofilm.
Figure 1. Experimental Set-Up. Mineral water was fed into the CFRABs
from 55 gallon drums, while acetate feeds were made in 7 L batches.
Biomass in
CFRAB (mg)
Table 1. Summary of the bacterial viability results for each run. The
mass of volatile solids per reactor slide, is provided along with the
live:dead ratio.
Denitrification Analysis
Methods
Antibiotic Conc.
(µg/L)
S0
0h
r
Nitrate contamination of drinking water
supplies due to agricultural runoff is a
prevalent problem, and it presents serious
health concerns. Low µg/L concentrations of
antibiotics have also been found in surface
waters worldwide. This research focuses on
how low concentrations of antibiotics affect
the rate of denitrification in slow rate
biofiltration systems which use biofilm to
treat drinking water.
• Nitrate sweeps were performed at the end of
the 7 day run by incrementally increasing the
nitrate concentration in the CFRAB.
nitrate (mg/L)
Introduction
34.4 ug/L (SMX and CIP)
45
50
55
60
65
70
75
References
80
Nitrate Substrate (mg/L)
Figure 6. Nitrate utilization in mg/L during each nitrate sweep. The highlighted
region of the graph represents a typical NO3- concentration range for untreated
drinking water (50 mg/L to 70 mg/L).
Kümmerer, K. (2009). Antibiotics in the aquatic environment--a
review--part I. Chemosphere, 75(4): 417-434.
Soares, M. I. M. (2000). Biological Denitrification of Groundwater.
Water, Air, and Soil Pollution, 123: 183-193.