Isolation of strains capable of mineralizing sulfamethoxazole ... - IWA

workkinkajouΒιοτεχνολογία

5 Δεκ 2012 (πριν από 4 χρόνια και 8 μήνες)

175 εμφανίσεις

(Initial page layout)



Isolation of strains capable of mineralizing sulfamethoxazole
from an acclimatized membrane bioreactor for wastewater
treatment



H. Bouju
*,
B. Ricken
*
, T. Wintgens*, P.F
-
X. Corvini*
,
***

and
B. Kolvenbach*



*
Institute for Ecopreneurship, School of Life Sciences, University of Applied Sciences Northwestern Switzerland,
40 Gründenstrasse, CH 4132 Muttenz, Switzerland
(E
-
mail:
helene.bouju@fhnw.ch
,
benjamin.ricken@fhnw.ch
,
thomas.wintgens@fhnw.ch
,
philippe.corvini@fhnw.ch
, boris.kolvenbach
@fhnw.ch)

**
Institute f
or Biology and Biotechnology of Plants, Westfälische Wilhelms University Münster, 48143 Münster,
Germany

*** School of the Environment, Nanjing University, 22 Hankou Rd., Nanjing, 210093, People's Republic of China




Abstract

In this study, we isolated f
ive strains capable of degrading ¬
14
C
-
labelled sulfamethoxazole to
14
CO
2

from a membrane bioreactor acclimatized to sulfamethoxazole, carbamazepine and
diclofenac. Of these strains, two belonged to the phylum of Actinobacteria
, while three are
members of the Proteobacteria.


Keywords



sulfamethoxazole; activated sludge; mineralization; strain isolation


INTRODUCTION

Antibiotics such as sulfamethoxazole (SMX) can be found at relatively high concentration in
surface waters worldwide. This causes concern due to the possibility of the spreading of antibiotic
resistances.

The removal of SMX in biological wastewater treatm
ent plants was reported to be highly
variable. Ozonation and ozonation
-
based advanced oxidation processes were reported to completely
transform SMX, yet they are energy
-
intensive and may release by
-
products that are more toxic than
the parent compound.Rece
ntly, two studies reported the biodegradation of SMX by pure strains
(Gauthier et al. 2010; Larcher and Yargeau 2011). However, the biodegradation rate remained
below 30% in both cases. This evidences the need to isolate and identify bacterial strains able

to
biodegrade SMX to higher extent.

From a membrane bioreactor (MBR) acclimatized with a synthetic effluent, mimicking real
wastewater, enriched with 100 µg/L of SMX, carbamazepine (CBZ) and diclofenac sodium (DF),
six strains able to grow on SMX as a car
bon and energy source

could be isolated by the serial
dilution technique.


MATERIAL AND METHOD

The lab
-
scale MBR
(Figure 1)
consisted of a 1.5 L glass reactor. The filtration unit consisted of 4
PVDF flat sheet membranes (Microdyn
-
Nadir GmbH, Germany) of 40 cm2 (4x10) each; with a
nominal pore size of 0.20

m. Filtration was run in sequence of 5 minutes filtration at a rate of 1.5
mL/min and 1 minute backwashing.Continuous air flow was supplied through a porous diffuser
made of sta
inless steel, and mixing was performed by a magnetic stirrer. The reactor was inoculated
with 1.1 L of sludge taken from the Sequencing Batch Reactor of ARA Birsfelden WWTP,
Switzerland. It was operated over 10 months at infinite SRT and a HRT of 12 hours
before biomass
sampling for enrichment cultures inoculation. At that time, the total solids concentration had an
average of 6 g/L, and the
average SMX removal rate was of

52 %.


Figure
1

Scheme of the laboratory scale membrane bi
oreactor

1
-

Bioreactor vessel; 2
-

Membrane plate modules; 3


Influent tank; 4


Effluent tank; 5


Influent peristaltic pump; 6


Air pump; 7


Level controller; 8


Digital pressure control; 9


Controlling valve; 10


Effluent peristaltic pump; 11


B
ackwashing peristaltic pump; 12


Reactor overflow; 13


Mono ethylene glycol flask for VOC trapping; 14


NaOH flasks for CO
2

trapping, 15


Digital pressure control; 16


Vacuum pump


Enrichment cultures were prepared in Erlenmeyer flasks containing 100
mL of mineral salts
medium (Stanier et al. 1966) containing 0.5 mM SMX as sole carbon source (MSX) and were
inoculated with 2 mL samples of biomass from the MBR before incubation at 28°C on a rotary
shaker at 130 rpm. Twice, after one month incubation each
, half of the culture volume was replaced
with fresh enriched MSX. After another month, 3 mL of enrichment culture was used to inoculate
100 mL of fresh MSX medium. A month later, the SMX degrading enrichment culture was diluted
in 0.85% (w/v) NaCl and pla
ted on Plate Count Agar (PCA, medium 464, DSMZ, Germany) and, to
possibly select for isolated fungi, Sabouraud agar (Sigma
-
Aldrich, Switzerland). Plates were
incubated overnight at 28°C, and six morphologically distinct types of colonies could be isolated.

Two colonies of each type were picked and isolated on PCA agar plates.

Homogeneous suspension of single colonies, and consortium were used to inoculate 20 mL of
MSX, in which 3nmol of
14
C
-
SMX

[aniline
-
14
C (uniformly)]

(Hartmann Analytic, Germany),

were
spiked. Mineralization was detected by trapping the formed
14
CO
2

in a 2 mL centrifugation tube
filled with 1 mL of 5M NaOH, fixed by copper wire in the headspace of the flasks, which were
hermetically sealed with rubber stoppers. All cultures were wra
pped in aluminium foil to prevent
photodegradation and incubated at 28°C on a rotary shaker at 120 rpm.

For mineralization of SMX by isolated strains, the latter were

grown on PCA plates for 48 hours
at 28°C and suspended in sterile physiological water (0.
85% (w/v) NaCl) to a final OD
600

of 1.0, of
which 500 µL were used to inoculate the Erlenmeyer flasks.



Fragments of 16S rRNA genes of the isolates mineralizing SMX were amplified by colony PCR
with universal 16S primers B27f and U1492r
(9)
. Amplified fragments were sequenced by Eurofins
MWG Operon (Germany) using 336r and 928f
(11)

and B27f and U1492r as sequencing primers.
Obtained sequences were subjected to nucleotide BLAST analysis.





RESULTS AND DISCUSSION

After 8 days of incubation,

first signs of mineralization were observed in the culture inoculated with
the enriched consortium, when an average of 0.3 ± 0.05 % (n=3) of the total applied radioactivity
was recovered in the 5M NaOH solution. The percentage of radioactivity recovered a
s
14
CO
2

increased gradually to reach a plateau at 58.0 ± 1.3 % af
ter 24 days incubation (Figure 2
).


Figure 2

Evolution of the mineralization rate of sulfamethoxazole in mineral medium enriched with 0.5 mM
14
C
-
labeled sulfamethoxazole inoculated with the
enriched consortium


As for the isolates, five out of six could mineralize SMX, because 24
-
44% of the total
radioactivity was found in the respective CO
2

traps after 16 days (
Bouju et al. 2012
).

None of the abiotic control showed accumulation of
1
CO
2
.



The identified strains belong to phyla Actinobacteria (
Microbacterium

sp. strain BR1,
Rhodococcus

sp. strain BR2) and Proteobacteria (
Achromobacter

sp. strain BR3 and
Ralstonia

sp.
strains HB1 and HB2). Although both
Ralstonia

strains were found to have id
entical 16S rRNA,
they possess different morphologies on PCA plates, as strain HB1 formed small, yellow colonies,
while strain HB2 formed larger, off
-
white colonies.


This is to our knowledge the first report of such high SMX mineralization in enriched cul
tures
and the first proof of mineralization of SMX by isolated strains, thus opening possibilities for a
better understanding of SMX removal in biological treatment of wastewater.


CONCLUSION

We investigated mineralization in an SMX
-
degrading enrichment cu
lture from which we later
isolated five strains able to mineralize SMX. This is to our knowledge the first report of such high
SMX degradation and actual proof of mineralization both in enriched cultures and in axenic strains.
Interestingly, cocultures of
Microbacterium sp. BR1 and Rhodococcus sp. BR2 showed both higher
mineralization rates and shorter lag times than cultures of the respective single strains, probably
caused by syntrophy. To a lesser extent, the same could be observed for samples containing

a
mixture of both Ralstonia isolates.

Currently, experiments are carried out to investigate possibly accumulating metabolites.



ACKNOWLEDGMENT

Project co
-
funding by the European Commission within the 7
th

Framework Programme under Grant
Agreement 265946 is acknowledged.



REFERENCES


Bouju, H., Ricken, B., Beffa, T., Corvini, P. F
-
X., Kolvenbach, B. (2012).
"Isolation of Isolation of
bacterial strains capable of mineralizing sulfamethoxazole from an
acclimated membrane
bioreactor."
Applied Environmental Microbiology
78
(1): 277
-
279
.

Gauthier, H., V. Yargeau, et al. (2010). "Biodegradation of pharmaceuticals by Rhodococcus
rhodochrous and Aspergillus n
iger by co
-
metabolism." Science of The Total Environment
408
(7): 1701
-
1706.

Larcher, S. and V. Yargeau (2011). "Biodegradation of sulfamethoxazole by individual and mixed
bacteria." Applied Microbiology and Biotechnology
91
(1): 211
-
218.

Stanier, R., N. Pal
leroni, and M. Doudoroff. 1966. The Aerobic Pseudomonads a Taxonomic Study.
Journal of General Microbiology
43
:159
-
271.