Impact of Antibiotics on Denitrifying Biofilm Bacteria
Jeremiah Rocha, Julie Swierenga, Professor David Wunder, Calvin College, Summer 2014
250
S inf,5
feed concentra tion (S inf)
S inf,4
200
150
S inf,2
100
DNA Analysis
Bacterial Viability
Biomark Fluidigm Real-time PCR
• Based on Live/Dead results, antibiotics do not
appear to have a significant effect on the
viability of the biofilm bacteria
• Biomass in the reactor is increased (compared to
control conditions) with 3.34 and 33.4 µg/L
antibiotics
• DNA was extracted from the biofilm using the
MoBio Power Biofilm DNA Extraction Kit
• Sample DNA was analyzed using the Biomark
Fluidigm Real-time PCR for different gene
markers present in denitrifying bacteria: 16S,
nosZ, nirS, nirK, norB, nrfA, napA, and narG
5h
r
4h
r
3h
r
2h
r
1h
r
S0
7 da ys
Run
Control
33.4 ug/L
3.34 ug/L
0.34 ug/L
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.
Objective
To understand the impact of low concentrations of
antibiotics on denitrifying bacterial biofilm used in
water treatment processes by assessing the
presence of relevant enzymes, denitrification rates,
and bacterial viability.
• Control runs contained no antibiotics in the feed
• Antibiotics were fed at three concentrations:
• 0.344 µg/L
• 3.34 µg/L
• 34.4 µg/L
• Antibiotics used: Sulfamethoxazole (SMX) and
Ciprofloxacin (CIP)
Methods
Experimental Set-Up
• Continuous-feed Rotating Annular Bioreactors
(CFRABs) were run for 7 days to simulate
biofiltration of drinking water.
• Mineral medium, acetate, nitrate, and antibiotics
were fed to the CFRAB which was seeded with
biofilm.
Viability Analysis
Volatile Solids Test
• Biofilm was harvested from the CFRAB and
combusted in an oven to determine volatile
solids, which represent biomass.
Conclusions
Figure 4. 96-Well Plate with
biofilm samples dyed with the
Live/Dead stain.
Results
Denitrification Rates
• Compared to the control and 0.34 µg/L
antibiotic feed conditions, denitrification rates in
water with environmentally-relevant NO3
concentrations (i.e., 50 mg/L to 70 mg/L) were
decreased by approximately 50% with water
containing 3.34 and 33.4 µg/L antibiotics
Nitrate Utilization Curves
7
6
5
Utilization rate (mg/L)
• The ratio of green to red fluorescence
represented the ratio of live to dead bacteria in
the biofilm.
nirS 1
22.91
19.67
20.46
--
Figure 3. Aluminum trays after
combustion in volatile solids analysis.
Live/Dead Stain
• 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.
nosZ
28.09
23.93
25.35
26.5
Table 1. DNA analysis results with the values for nosZ and nirS1
represented in a relative Evagreen dye fluorescence expression count
Denitrification Analysis
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
Preliminary DNA Analysis
• The following genes are present in the DNA for
all feed conditions: nirK2, nirS1, nirS2 nosZ and
are appear to be affected by antibiotics
S3
S2
S1
SF inf,0
S5
S4
S inf,1
75
50
S inf,3
effl uent concentra tion (S)
0h
r
Nitrate contamination of drinking water supplies,
partially attributable to agricultural runoff, is a
prevalent problem. Nitrate presents public health
concerns, especially for pregnant mother and
infants. 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 biological denitrification
in biofilm bacteria used to treat drinking water
supplies.
nitrate (mg/L)
Introduction
• Nitrate sweeps were performed at the end of
the 7 day run by incrementally increasing the
nitrate concentration in the CFRAB.
Control 1 (no antibiotics)
4
3.44 ug/L (CIP and SMX)
33.4 ug/L (CIP and SMX)
3
• 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.
• Some denitrification genes were present in
the biofilm DNA and were affected by
antibiotic concentration.
Acknowledgments
Jansma Family Research Fund
Mr. Bob De Kraker, Calvin College
Mr. Rich Huisman, Calvin College
Mr. Phil Jasperse, Calvin College
Ms. Lori Keen, Calvin College
Mr. Randall DeJong, Calvin College
Mr. Scott Prentice, Calvin College
0.334 ug/L (CIP and SMX)
Control 2 (no antibiotics)
33.4 ug/L (CIP and SMX)
2
References
1
0
40
45
50
55
60
65
70
75
80
Nitrate Substrate (mg/L)
Figure 1. Experimental Set-Up. Mineral medium was fed into the CFRABs
from 55 gallon drums, with acetate and antibiotics were feed separately.
Figure 5. 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
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Soares, M. I. M. (2000). Biological Denitrification of Groundwater.
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