APPLIED MICROBIAL AND CELL PHYSIOLOGY
Characterization of extracellular polymeric substances from biofilm in the process of starting-up a partial nitrification process under salt stress
Zhao-Ji Zhang &Shao-Hua Chen &Shu-Mei Wang &Hong-Yuan Luo
Received:15June 2010/Revid:9October 2010/Accepted:10October 2010/Published online:30October 2010#Springer-Verlag 2010
Abstract In this study,the characteristics of extracellular polymeric substance (EPS)fractions of biofilm during the process of establishing a partial nitrification under salt stress were analyzed in terms of concentrations,molecular weight distribution,and three-dimensional excitation –emis-sion matrix (EEM)fluorescence spectroscopy.A partial nitrification was formed successfully with a salinity of 1%.Results indicated that the amount of total EPS incread from 54.2mgg −1VSS −1on day 1to 99.6mgg −1VSS −1on day 55due to the NaCl concentration changed from 0to 10.0gL −1in a biofilm reactor.The changes of looly bound EPS (LB-EPS)compounds under different salt concentrations appeared to be more significant than tho of the tightly bound EPS.A clear relea of polysaccharides in the LB-EPS fraction was detected during the enhance-ment of salinity.This was c
onsidered as a protective respon of bacteria to the salinity.Three fluorescence peaks were identified in the EEM fluorescence spectra of the EPS fraction samples.Two peaks were assigned to the protein-like fluorophores,and the third peak was located at the excitation/emission wavelengths of 275nm/425–435nm of the spectra of EPS fractions till the salinity maintained constant at 1%.This information is valuable for understanding the characteristics of EPS isolated from biomass in a saline nitrogen removal system.
Keywords Extracellular polymeric substances .Salinity .Partial nitrification .Excitation –emission matrix
Introduction
Recently,concentrated ammonium waste streams contain high strength of salts from some coastal wastewater treatment plants (WWTPs)has attracted great attentions by many rearchers and engineers due to the low nitrogen removal efficiency by nitrification –denitrification biological nitrogen removal (BNR)process (Windey et al.2005;Mosquera-Corral et al.2005).Previous rearches indicated that salt stress has a negative impact on BNR performance (Panswad and Anan 1999a ).However,fresh activated sludge could be adapted to saline wastewater by increasing salt concentra
tion slowly (Dahl et al.1997)and the nitrogen removal rate could even be improved when eded with salt endurable cultures (Panswad and Anan 1999b ).Further-more,partial nitrification,in which the nitrite-oxidizing bacteria (NOB)are inhibited during the nitrification process,has been reported as a promising and sustainable technique in the BNR process even in a saline system (Mosquera-Corral et al.2005).The partial nitrification could be achieved bad on a strategy of pH control when the salt concentration was as high as 425mM (Mosquera-Corral et al.2005).Unfortunately,the adaptability of activated sludge to the salinity in the partial nitrification system has not been well investigated.
Among all the possible reasons,production of extracellular polymeric substances (EPSs)from activated sludge may play a significant role in protecting some microorganism from salt stress.In general,the EPS is a complex mixture of proteins,polysaccharides,lipids,DNA,and humic acid substances that surround cells and creates a matrix of microbial flocs and films (Liao et al.2001).The production of EPS has been taken as a crucial way to protect cells against the adver environ-ment in wastewater treatment system,such as changes in
Z.-J.Zhang :S.-H.Chen (*):S.-M.Wang :H.-Y .Luo Key Laboratory of Urban Environment and Health,
Institute of Urban Environment,Chine Academy of Sciences,Xiamen 361021,People ’s Republic of China e-mail:shchen@iue.ac
Appl Microbiol Biotechnol (2011)89:1563–1571DOI 10.1007/s00253-010-2947-y
organic loading rate,carbon and nitrogen sources(Yang
and Li2009).The EPS compounds relead in the
bioreactor,such as bacterial exopolysaccharides have been
considered as a protective respon of bacteria under salt
stress(Sheng and Yu2006).The amount of total EPS
extracted from the halotolerant bacterium Rhodopudo-
monas acidophila(Sheng and Yu2006)and the medicinal
mushroom Phellinus linteus(Zou et al.2006)incread
concomitantly with salt concentration.
Hence,in a saline partial nitrification process,it can be
inferred that the EPS fractions content in the activated
sludge may shift as a respon to the osmotic pressure
caud by the salinity during the start-up of a partial
nitrification process in the prence of salts.However,to
the best of our knowledge,there is a lack of rearch about德的组词
the dynamic of EPS fractions in a BNR system under salt
stress.In the prent work,the aim is,therefore,to
investigate the properties of EPS fractions extracted from
biomass in the cour of establishing a saline partial
nitrification process.
Materials and methods
Start-up of a partial nitrification under salt stress
A pilot-scale quencing batch biofilm reactor(SBBR),
which operated for55days,was ud to start-up the partial
nitrification under salt stress.The working volume of the
SBBR was6.5L,with an exchange volume ratio of50%.
Non-woven porous polyester was ud as a biomass carrier,
and the packing rate was40%(v/v).The temperature was
fixed at30−35°C during the whole experiment by means of
a thermostat jacket.Mixing inside the reactor was
performed by a mechanical stirrer,and the air was supplied
via four diffurs at the bottom of the reactor.
The SBBR was operated in intermittent aeration cycles
of4h(HRT=2days)in order to avoid possible toxicity of
nitrite accumulation under salt stress.The reactor was fed
with a synthetic autotrophic medium described by Van de
Graaf et al.(1996).NaCl concentration was gradually
incread from2.0to10.0gL−1over44days and then kept
constant at10.0gL−1for the duration of the study.The
feeding solution was diluted with tap water,and the
nitrogen loading rate(NLR)was incread to0.093kg
NH4+–Nm−3day−1gradually over the next25days.Acti-vated sludge from an aeration tank in a continuous WWTP
(Xin lin,Xiamen,China)was ud as inoculum of the
nitrifiers(VSS 1.746gL−1).The SBBR system had
operated for2weeks before the start of the experiment,
and the synthetic NLR was kept as low as the influent of
the WWPT due to achieving a new steady EPS concen-
trations caud by substrate and environment changes.EPS fractionation protocol
Prior to fractionation,six biofilm samples were obtained
from the supporting fabrics by a razor blade with different
salinity as scheduled,5ml of each biofilm sample was
conducted in triplicate for EPS fractionation.The proce-
dures of EPS fractionation are described in detail by Yu et
al.(2009).In brief,the collected biofilm ttled at4°C for
1.5h and were re-suspended in an EPS buffer solution
described by Frølund et al.(1995).For the next step,
biofilm samples were centrifuged at2,000×g for15min,re-
suspended again with EPS buffer solution,and centrifuged
at5,000×g for15min.The organic matter in this bulk
solution was considered as the LB-EPS.The diments
were re-suspended again and extracted using ultrasound
(20kHz and480W)for10min,and the bulk solutions
were centrifuged at20,000×g for20min.The organic
天空之神
matter in this bulk solution was considered as the tightly
bound EPS(TB-EPS).All EPS fraction samples were
filtered through polyvinylidene fluoride membranes with a
pore size of0.45μm before measurement.
Analytical methods
The amount of proteins(PN)in the EPS fractions was
determined by the Bradford method(Bradford1976)using
bovine rum albumin as the standard.Polysaccharides(PS)
情绪abc
were measured by the Anthrone method(Gaudy1962)using
gluco as the standard.Humic acid was detected by a
Modified Lowry Method Kit(Bioteke,China)using humic
acid(Sigma,USA)as the standard(Liu and Fang2002).The
amount of EPS was measured as the sum of the three
substances,standard deviations are given for three repetitions
each.Total organic carbon(TOC)was measured by a
Shimadzu analyzer(TOC-V,Shimadzu,Japan).Zeta poten-熟石灰化学式
tial of the fractions was quantified by a Malvern Zetasizer
(Nano Z,Malvern,UK).The pH and DO of the bulk solution
were detected by a Hach analyzer(HQ40d18,Hach,USA).
The concentrations of NH4+–N,NO2−–N,and NO3−–N were measured by a flow injection analyzer(8500Series2,
QuickChem,USA).Total nitrogen(TN)was measured by a
Shimadzu analyzer(TNM-1,Shimadzu,Japan).Concentra-
tions of mixed liquor suspended solids and mixed liquor
volatile suspended solids were measured as described in the
standard method(NEPA Chine2002).
Bacterial populations of ammonia-oxidizing bacteria
(AOB)and NOB were identified by fluorescence in situ
hybridization(FISH)technique.Biofilm was collected,
washed,and fixed in4%paraformaldehyde solution.The
process of in situ hybridization was carried out according to
the study of Amann et al.(1990).Biofilm samples
simultaneously hybridized with NEU653(5′-CCC CTC
TGC TGC ACT CTA-3′)(Wagner et al.1995)and Nit3(5′-
CCT GTG CTC CAT GCT CCG-3′)(Daims et al.2000),which belong to the genera Nitrosomonas and Nitrobacter ,respectively,labeled from Invitrogen Corporation (California,USA).All fluorescence signals were recorded with a LSM 710confocal lar scanning microscope (CLSM;Carl Zeiss,Germa
ny),and the CLSM images were analyzed by the standard software for the LSM 710.
Molecular weight distribution analysis
The fractionation of the LB-EPS and TB-EPS components was achieved by filtration of the samples through the regenerated cellulo membrane,which have nominal weight cutoff values of 30,10,3,and 1kDa (PLTK,PLGC,PLBC,and PLAC,Millipore,USA).The filtration took place in a cylindrical dead-end stirred cell (Amicon 8010,Millipore,USA).The cell was stirred at a speed of 500rpm with a magnetic stirrer (RTC basic,IKA,Germany)at room temperature.The air pressure was maintained constant at 100kPa by a fine-pressure reducing valve (Tescom,USA)at each membrane filtration.Both LB-EPS and TB-EPS filtered samples were analyzed for non-purgable organic carbon concentrations using a TOC analyzer (TOC-V wp ,Shimadzu,Japan)to calculate the molecular weight (MW)portions.Three-dimensional excitation –emission matrix fluorescence spectroscopy
Fluorescent fingerprint,known as three-dimensional exci-tation –emission matrix (EEM),has been confirmed as a
010********
60
7080
Time(d)
m g N L -1
0102030405060
708090100N A R (%)
Fig.1Concentrations of nitrate and nitrite of the effluent.The NAR
was calculated as follows:NAR %ðÞ¼NO À2
NO À2
þNO À3
Â100%
a
b
c
d
Fig.2CLSM images of in situ hybridization of the biofilm with a FITC-labeled NEU653(green )and b Cy 3-labeled Nit3(red )on day 50;c biofilm background;d simultaneous hybridization of the biofilm with NEU653and Nit3on day 50
uful technique to investigate the organic substances prent in different solutions due to its high lectivity and non-destruction of samples (Baker 2001;Chen et al.2003;Aryal et al.2009).The fluores
cence property of organic substances in the liquid can be obtained by simultaneously changing excitation and emission wave-lengths.As a fast and nsitive technique,EEM has been ud to characterize the EPS compounds on membrane bioreactor fouling (Wang et al.2009),to monitor the dissolved organic matter for recycled water system (Henderson et al.2009),and to access the EPS chemical composition of aerobic and anaerobic sludge (Sheng and Yu 2006;Ni et al.2009).
All three-dimensional EEM spectra were measured using a luminescence spectrometry (F-4600FL Spectro-photometer,Hitachi,Japan).In this study,the EEM spectra were collected with corresponding scanning emission spectra from 200to 500at 5nm increments by varying the excitation wavelength from 200to 400nm at 5nm sampling intervals.Excitation and emission slits were kept at 10nm and the scanning speed was maintained at 1,200nmmin −1.The spectrum of deionized Milli-Q water (Millipore,USA)was recorded as the blank.All the EEM data,which plotted as the elliptical shape of contours,were procesd by the software Origin 7.0(OriginLab Inc.,USA).Statistical analysis
The nonparametric,distribution-free,rank statistic propod by Spearman was adopted to evaluate the relationship between two variables at a 95%confidence level (Li and Yang.2007).The Spearman rank correlation coefficient,R s,is a measure of statistical dependence between two variables and it asss how well the relationship between two variables can be described using a monotonic functi
on.It is a strong correlation between the variables when the R s higher than a Spearman ’s critical value,otherwi,the two variables are not correlative.
Results
Start-up a partial nitrification under salt stress
A steady production of EPS had been obrved after 2weeks of adaptation operation.After that,NaCl began to be introduced to the reactor.The profile of the nitrite accumulation rate is illustrated in Fig.1.The NaCl concentration in the SBBR was incread stepwi to 10.0gL −1over 44days.As Fig.1shows,an increa of NaCl concentration from 4.5to 6.5gL −1(day 21)results in a sudden increa of nitrite accumulation rate (NAR).From this day onwards,the mean NO 2−–N accumulation rate reached over 80%,suggesting that an effective NOB
sludge
Samples
P N (m g g -1V S S -1)
P S (m g g -1V S S -1)
H A (m g g -1V S S -1)
sludge
Samples微信推广文案
N a C l (g L -1)
可乐鸡腿N a C l (g L -1)
大学生就业现状N a C l (g L -1)
LB-EPS
LB-EPS a
b
c
Fig.4Variations of proteins (a ),polysaccharides (b ),and humic acid (c )levels in the LB-EPS and TB-EPS fractions with salinity.Error bars reprent standard deviation,n =3
102030405060708090100110120Seeding sludge
1d Samples
E P S (m g g -1
V S S -1)
1234567891011N a C l (m g L -1
)
8d 18d 33d 55d
Fig.3Variation of total EPS in the biofilm with salinity
inhibition was achieved under salt stress.The mean TN removal rate was0.012kgNm−3day−1.The distribution of AOB and NOB in the biofilm was analyzed by the FISH technique.CLSM images of the in situ hybridized samples show that AOB were mainly located on the surface of the biofilm,whereas the NOB were found to exist in the internal layer of the biofilm(Fig.2).
Variations of EPS fractions under salt stress
小米笔记本电脑官网The experimental results related to the concentrations of total EPS with salinity are shown in Fig.3.It is clear that the amount of the total EPS in terms of protein,polysac-charide,and humic acid concentrations was incread from 54.2mgg−1VSS−1on day1to99.6mgg−1VSS−1on day55 by gradually increasing the NaCl concentration from2.0to 10.0gL−1.Percentages of PN,PS,and HA content in the total EPS were36.6%,37.4%,and26.0%on day1, respectively.Afterwards,percentages of PS in the total EPS were incread to50.8%on day18when the NaCl concentration was incread to5.5gL−1.The concentration of PS incread from20.3mgg−1VSS−1on day1to 59.7mgg−1VSS−1on day55relative to the increa in salinity.The results indicated that salinity has a signifi-cant impact on the polysaccharide levels in total EPS for the NaCl concentration fluctuating in the range of2.0to 10.0gL−1.
Variations of PN,PS,and HA in LB-EPS and TB-EPS fractions relative to salinity are show in Fig.4.It is obrved that the PN occurred mainly in the TB-EPS fraction on day1and no clear changes of proteins in LB-EPS and TB-EPS fractions have been monitored in this experiment.Nevertheless,the results were different with respect to the PS and HA content in the EPS fractions.Most of the PS occurred in the TB-EPS on days1and8,above 95%.A clear increa of PS in the LB-EPS fraction was detected when the NaCl concentration incread to5.5and 9.0gL−1on days18and33,respectively.At the last 2weeks,the salinity was kept constant at10gL−1,the relea of LB-EPS fraction was faded away.The situation of HA in the EPS fractions was similar to that of PS.MW distribution of the EPS fractions
As mentioned above,both LB-EPS and TB-EPS fractions were fractionated into components with different MWs in order to investigate the relationship of molecular distribu-tion of the EPS fractions with salinity.It was noted that the different EPS fractions had a heterogeneous MW distribu-tion with different salinity.The dominant fraction of LB-EPS was that of small molecules with MW<1kDa on days1and8(Table1).A clear relea of macromolecules, which have a MW of0.45μm−30kDa,was obrved in the LB-EPS fraction on day18.Such differences may be attributed to relea of an increment of polysaccharides from the bacteria or plasmolysis caud by the prence of salinity.Th
e majority of TB-EPS fraction was mainly compod of macromolecules having a MW of0.45μm–30kDa and small molecules(<1kDa),with a minor percentage of other molecules,exhibiting a bimodal character during the whole experiment.
EEM fluorescence spectra of the EPS fractions
The EEM fluorescence spectra of LB-EPS and TB-EPS fractions under different salt stress are illustrated in Fig.5 and the Zeta potential and pH values are listed in Table2. As discusd above,each EEM fluorescence spectrum gave information about the chemical composition of EPS samples.Three main peaks could be obrved from the fluorescence spectra of LB-EPS and TB-EPS on day18. Peak A was located at the excitation/emission wavelengths (Ex/Em)of225–230/330–340nm,while Peak B was obrved at the Ex/Em of275–280/330–340nm.The two peaks were assigned to the reported aromatic protein-like substances(Peak A)and tryptophan protein-like substances (Peak B)(Chen et al.2003;Sheng and Yu2006).The location of Peak B in the EPS fraction samples showed a blue shift in terms of emission wavelengths relative to the fluorescence peak location of proteins reported previously (Ex/Em of276–281/340–370nm)(Baker2001).The third peak(Peak C)located at the Ex/Em of275/425–435nm, which corresponds to the humic acid-like substances(Chen
Table1Percentages of chemical components content in LB-EPS and TB-EPS with various molecular fractions
Percentage(%)1day8days18days33days55days
LB-EPS TB-EPS LB-EPS TB-EPS LB-EPS TB-EPS LB-EPS TB-EPS LB-EPS TB-EPS
0.45um−30kDa10.3844.278.0832.2843.7644.1428.4028.0718.3932.85 30kDa−10kDa 2.36 5.92 5.38 5.12 3.68 5.998.98 4.577.27 2.06 10kDa−3kDa 4.19 2.67 2.187.32 4.38 3.97 6.69 3.2710.29 1.79 3kDa−1kDa32.647.8828.99 1.18 5.388.9820.127.27 5.72 4.39 <1kDa50.4339.2655.3754.1042.8042.6735.8156.8258.3358.90
