University of Warmia and Mazury in Olsztyn

FACULTY OF ENVIONMENTAL !OTECTION AN" FI#$EIE#

La%&e'ort(
"etermination of t)e effe*tiveness of +aste+ater treatment %y
a*tivated slud,e
and
Fluores*ent in situ )y%ridization -FI#$.
/rou' 0( Treated +aste+ater sam'le ta1en at 2345647522
Made by:
To%ias 8os*)
#as*)a $eilemann
e%e**a 9u)n
Lecturer:
!)" 9atarzyna 8ernat
"r4 Ma,dalena :ielins1a
Date: 28.05.2011
2
Determination of the effectiveness of wastewater treatment by activated sludge
Contents
1. im and sco!e of study..........................................................................................................."
2. #esults.....................................................................................................................................5
". Discussion.............................................................................................................................12
nalytical Methods in $iological %ystems
"
Determination of the effectiveness of wastewater treatment by activated sludge
24 Aim and s*o'e of study
&he aim of this laboratory was to determine the effectiveness of !ollutants removal by
activated sludge in de!endence on '(D)* ratio in wastewater. ctivated sludge is a !rocess
used to treat sewage and industrial wastewaters by using an aeration tan+. ,n this e-!eriment
two laboratory single.stage aeration tan+s with activated sludge were used. &he tan+s had the
same volume/ the same !um! with same flow rate and the same dissolved o-ygen
concentration. &here were two different variants used. 0ariant 1 had a low '(D)&1* ratio
and variant 2 had a high '(D)&1* ratio. &he '(D)&1* ratio s!ecifies the effect of nitrite
and nitrate as inhibitors. &he scheme of this e-!eriment can be seen in 2igure 1.
Fi,ure 2( #*)eme of t)e e;'eriment4 <( flo+ rate in dm=>)? V( rea*tor volume dm=? Ci( 'ollutants
*on*entration influent m, O7>dm=? Ce( 'ollutants *on*entration effluent m, O7>dm=? @( mass of a*tivated
slud,e m,T#>dm=? W( ra+ +aste+ater? TW( treated +aste+ater
2or activated sludge/ the following !arameters were determined:
flow rate for the aeration tan+s
reactor volume
mass of activated sludge in aeration tan+s
%0, 3 sludge volume inde-
nalytical Methods in $iological %ystems
4
Determination of the effectiveness of wastewater treatment by activated sludge
ll of the ne-t !arameters were determined for both variants of treated wastewater:
!5
'(D
&1*
*.*54
*.*(2
*.*("
2or raw wastewater only the !5/ '(D/ &1*/ and *.*5
4
from the second variant was
measured.
(n another laboratory day/ a fluorescent in situ hybridi6ation was !erformed/ to get the
!ercentage of ammonium o-idi6ing bacteria 7($8 in the activated sludge tan+s. &he tan+s
were the same as in the e-!eriment before and visuali6ed in 2igure 1. 2irst sam!les from the
two tan+s are ta+en and fi-ed. fterwards they were hybridi6ed and then evaluated via
microsco!e and image analysis.
nalytical Methods in $iological %ystems
5
Determination of the effectiveness of wastewater treatment by activated sludge
74 esults
"etermination of t)e effe*tiveness of +aste+ater treatment %y a*tivated slud,e
,n &able 1 are the measured values of the laboratory determinations. &he !5 was measured
with a standard !5.meter. '(D was determined with dichromate method. $(D was
calculated with the '(D. ,n raw water it is assumed that the $(D is 90 : of the '(D/ in
treated water 25 : of '(D were assumed. &he &1* sam!les were minerali6ed and
determined with the distillation)titration method. mmonium in raw water was measured via
distillation)titration method and in treated water via the nessleri6ation. *itrite and *itrate
were determined with the standard s!ectro!hotometric methods/ with a wavelength of 410 nm
for *itrate and 520 nm for *itrite. &he values with grey colour are the mean values for all
grou!s. &hey were used because too high values were determined with our measurement.
Ta%le 2( "etermined values of t)e +aste+ater sam'les? ,rey values are mean values from all ,rou's
a+ Treated
"etermination Unit V2 V7 V2 V7
'$ !5 8/4 8/9 8/"
CO" mg (
2
)dm
"
548 519/0 124/0 152/0
8O" mg (
2
)dm
"
"28/8 "0;/9 "1/0 "8/0
T9N mg &1*)dm
"
58/25 199/9 0/0
Ammonia mg *.*54)dm
"
4</49 1""/5 0/2<8 2/110
Nitrite mg *.*(
2
)dm
"
. . 0/280 0/025
Nitrate mg *.*(
"
)dm
"
. . 28/0 "2/9
=>uations to calculate the values from &able 1:
8 l ) mg 7
0
1000 ? 8 ? n ? 8 b . a 7
@ '(D
'r
'hemical o-ygen demand 7dichromate method8
a: volume of MohrAs salt used for blan+ 7ml8
b: volume of MohrAs salt used for sam!le 7ml8
0: volume of sam!le 7ml8
8: factor converting obtained result to mg of (
2
n: normality of MohrAs salt 70/05 *8
nalytical Methods in $iological %ystems
9
Determination of the effectiveness of wastewater treatment by activated sludge
8 l ) mg 7
0
1000 ? b8 . a 7 ? 14 / 0
@ &1* &otal 1Beldahl nitrogen
0/14: milligrams of nitrogen corres!onding to 1 ml 0/01 * 5'l
a: volume of 0/01 * 5'l in =rlenmeyer flas+ 7ml8
b: volume of 0/01 * *a(5 used during sam!le titration 7ml8
0: sam!le volume 7ml8
8 l ) mg 7
0
1000 ? a ? =
@ mmonia mmonia 7nessleri6ation method8
a: coefficient from standardi6ation curve 70/25<8
=: absorbance of sam!le
0: sam!le volume 7ml8
8 l ) mg 7
0
1000 ? b8 . a 7 ? 14 / 0
@ mmonia mmonia 7distillation method8
0/14: milligrams of nitrogen corres!onding to 1 ml 0/01 * 5'l
a: volume of 0/01 * 5'l in =rlenmeyer flas+ 7ml8
b: volume of 0/01 * *a(5 used during sam!le titration 7ml8
0: sam!le volume 7ml8
8 l ) mg 7
0
f ? 1000 ? a ? =
@ *itrate *itrate
a: coefficient from standardi6ation curve 70/2"8
=: absorbance of sam!le
0: sam!le volume 7ml8
2: dilution factor
8 l ) mg 7
0
1000 ? a ? =
@ *itrite *itrite nitrogen
a: coefficient from standardi6ation curve 70/0"498
=: absorbance of sam!le
0: sam!le volume 7ml8
nalytical Methods in $iological %ystems
<
Determination of the effectiveness of wastewater treatment by activated sludge
&able 2 shows the results for the total solids and sludge volume inde- in activated sludge from
variant 1 and 2.
Ta%le 7( "etermination of T# and #VI
"etermination Unit V2 V7
T# mg &%)dm
"
2;"4 2<;0
#VI cmC)g 11< 12"
&he e>uations used to calculate the values from &able 2 are the following:
&otal solids of activated sludge 7&%8
a: filter weight with residues after drying
7g8
b: filter weight 7g8
0: sam!le volume 7cmC8
8 g ) C cm 7
&%
10 ? 0 ? 1000
@ %0, %ludge volume inde- 7%0,8
0: the volume of settle able solids after "0 minutes of settlement 7cmC8
&%: total solids concentration 7mg &%)l8
&able " shows us the su!!lementary calculations which had to be done for the further
calculations in &able 4. &he used formulas are the following:
*%yn &1* / e &1* / i *(-
' . ' . ' @ '
mmonia concentration o-idi6ed by activated sludge
* '(D / e '(D / i *%yn
2 ? 8 ' . ' 7 ? D @ '
mmonia concentration used for biomass synthesis
3 2 *( * / e *( * / e *(- *red
' . ' . ' @ '
*itrogen concentration due to denitrification
'
i/&1*
is the &1* concentration in the influent
'
e/&1*
is the &1* concentration in the effluent
D is the biomass yield
'
i/'(D
is the organics concentration in the influent
'
e/'(D
is the organics concentration in the effluent
2
*
is the nitrogen content
nalytical Methods in $iological %ystems
8 l ) &% mg 7
0
1000 ? 1000 ? 8 b . a 7
@ solids &otal
8
Determination of the effectiveness of wastewater treatment by activated sludge
'
e/*.*(2
is the nitrite concentration in the effluent
'
e/*.*("
is the nitrate concentration in the effluent
2or $(D)&1* from 1 to 5: D@0/" g '(D)g '(D
2or $(D)&1* from 9 to 10: D @0/5 g '(D)g '(D
2
*
@ 0/05 g *)g s.m.
Ta%le A( Cal*ulations to determine t)e effe*tivenessBs
#u''lementary Cal*ulations
'
*(-
mg *(
E
)dm
"
4</95 191/14
D g '(D)g '(D 0/5 0/"
2
*
g *)g %.m. 0/05 0/05
'
*syn
mg *.*54)dm" 10/9 5/49
'
*red
mg *
red
)dm
"
1;/"< 128/52
,n the table below are the calculations for the effectivenessAs. &he flow rates were read from
the !um! and the si6e of the reactor was measured to determine the volume. fterwards you
can calculate the retention time:
>
0
@ t
&he organic loads were calculated via:
0
' ? >
@ F
i
&he sludge loading rate can be calculated
with
E ? 0
' ? >
@ F
i
E
&he effectiveness of organics removal is calculated with 100 ?
'
' . '
@
i
e i

&he hydraulic loading rate can be determined by

0
? >
@ 5L#
24
=ffectiveness of nitrification:
100 ?
' . '
'
@
*%yn &1* / i
*(-
ion nitrificat

=ffectiveness of denitrification: 100 ?

'
'
@
*(-
*red
ation denitrific

ll symbols used in the formulas can be found in the table below or are already described
before.
nalytical Methods in $iological %ystems
;
Determination of the effectiveness of wastewater treatment by activated sludge
Ta%le C( Cal*ulations of t)e effe*tivenessBs
"etermination Unit V2 V7
CO">T9N . ;/41 "/10
Flo+ rate > dm
"
)h 0/18 0/18
ea*tor volume 0 dm
"
9/"4 9/"4
etention Time t
#
h "5/22 "5/22
Or,ani* load CO" F
'(D
+g '(D)m
"
d 15/59 14/95
#lud,e loadin, rate F
E/'(D
+g '(D)+g &% d 5/"0 5/25
Or,ani* load 8O" F
$(D
+g $(D)m" d ;/"4 8/<;
#lud,e loadin, rate F
E/$(D
+g $(D)+g &% d "/18 "/15
$ydrauli* loadin, rate 5L# mC)m
"
d 0/98 0/98
Effe*tiveness or,ani*s removal
CO"
G
'(D
: << <1
Effe*tiveness or,ani*s removal
8O"
G
$(D
: ;1 88
Effe*tiveness nitrifi*ation G
nitrification
: 100 100
Effe*tiveness denitrifi*ation G
denitrification
: 41 80
&he volume of the tan+ was determind with the following values in the figure:
2igure 2 you can see dimensions of the
aeration tan+ and with the +nowledge of the
de!th of 18 cm you can calculate the volume
of the tan+.
nalytical Methods in $iological %ystems
Fi,ure 7( dimesions of t)e aeration tan1
10
Determination of the effectiveness of wastewater treatment by activated sludge
Fluores*ent in situ )y%ridization
2or the determination of the !ercentage of ($s in the sam!les/ 14 sam!les were e-amined
via the ,mage analysis. &he !rinci!le of this analysis is to measure the biovolume fraction of
the ($s relative to the total microbial community. &he ($ area fraction is then measured
as !ercentage of total area of all organisms. &he result of this analysis is shown in &able 5.

Ta%le 6( "etermined values and mean values of t)e determination of AO8s in t)e sam'les
Determined !ercentage of ($s in the
sam!le 7:8
#eactor 1 #eactor 2
11/99 11/84
9/<8 8/;1
29/00 19/50
10/42 45/14
29/10 59/40
</25 19/;0
2"/5< 90/<<
Mean value 7:8
0/19 "1/00
2or the gra!hical demonstration of the effect of the '(D)* value for effectiveness of
nitrification and !ercentage of ($s in the reactor shown in 2igure " the values from &able 9
were used.
Ta%le 0( Values used for ,ra')i*al demonstration
#ea+tor 1 #eactor 2
=fficiency of
nitrification 7:8 100 100
'(D)* ;/41 "/10
($ 7:8 19 "1
nalytical Methods in $iological %ystems
11
Determination of the effectiveness of wastewater treatment by activated sludge
3,1 9,41
0
5
10
15
20
25
30
35
0
10
20
30
40
50
60
70
80
90
100
*itrification =fficiency and ($7:8 versus '(D)*
'(D)* ratio

(
$

7
:
8
*
i
t
r
i
f
i
c
a
t
i
o
n

e
f
f
i
c
i
e
n
c
y

7
:
8
Fi,ure A( Im'a*t of CO">N ratio to 'er*enta,e of AO8 'resent and effi*ien*y of nitrifi*ation
Hictures of the total microbial community and the ($s !resent can be seen in 2igure 4 and
2igure 5. &he !ictures are showing the sam!les under the e!ifluorescence microsco!e.
Fi,ure C( !i*ture of total mi*ro%ial *ommunity
'resent in a*tivated slud,e sam'le under
e'ifluores*en*e mi*ros*o'e
Fi,ure 6( !i*ture of AO8s -dar1 red. 'resent in
a*tivated slud,e sam'le under e'ifluores*en*e
mi*ros*o'e
nalytical Methods in $iological %ystems
($

12
Determination of the effectiveness of wastewater treatment by activated sludge
A4 "is*ussion
&he two reactors are different in the '(D)&1* ratio. ,n reactor 1 the ratio '(D)&1* is with
a value of ;/4mg)l com!ared with "/1 mg)l about "times higher. &he big difference is caused
by the &1* value which is in variant 1 with a value of 58/25 mg)l and in variant 2 with an
value of 199/9 mg)l around three times smaller. &he organic load '(D/ the sludge loading
rate and the organic load $(D are almost e>ual in both reactors.
&he &% value of variant 1 was 2;"4 mg)l. &he &% value of variant 2 was 2<;0 mg)l. &he %0,
were with 11< cm
"
)g 7variant 18 and 12" cm
"
)g 7variant 28 almost e>ual but this values are
trendly too high. ,deally the %0,.value should be in the range of 50 to 100 mL)g for most
conventional activated sludge !lants Ihtt!:))www.wrights.
trainingsite.com)ct%lud1onb.htmlJ. (ur sludge is s!ecified as filament growth.
&he results of the nitrification !rocess or the ammonia concentration in treated wastewater is
with values of 0/2<8 7#18 and 2/11 7#28 very low. &hat means that almost all mmonia was
converted into nitrate via nitrite. &herefore you can see that the conversion was done
com!letely. &his result was a!!roved by the calculation of the effectivness in nitrification
where our calculated values are 100: in both reactors. %o the amount of bacteria in the
aerobic tan+ were able to convert the ammonia. ,n reactor 1 we determined 19: of nitrifying
bacteria with the 2,%5 method. ,n reactor 2 the evaluated amount was around "1: nitrifying
bacteria of the whole community. $ut this method/ esc!ecially the image analysis is a method
with a high standard deviation. &his comes from the subBective selection of the area of all
microorganismns and the target selection of the itrifying bacteria. &he ratio of both amount of
selected microorganismns 7com!are figure "8 is the amount of bacteria which are res!onsible
for the nitrification !rocess. Ke did a mean value of seven measurements.
Lee studied similar e-!eriment/ where analy6ed microbial community from activated sludge
which characteri6ed high .nitrate .removing and the second one low. nitrate .removing. &he
number of nitrifying bacteria was higher in the low.nitrate removal reactor 7'#.,,8 than in
high.nitrate removal reactor 7'#.,8/ and nitrifying bacteria such as Nitrospira sp. and
Nitrosomonas sp. were dominant in the low.nitrate removal reactor. Lee maintain/ that
anaerobic ammonium o-idation and nitrate o-idation may occur in low nitrate removing
sludge and inhibit denitrification I2J.
nalytical Methods in $iological %ystems
1"
Determination of the effectiveness of wastewater treatment by activated sludge
&he nitrifying bacteria in the activated sludge grow in the form of micro colonies embedded
in floc+ of heterotro!hic microorganisms/ ma+ing the heterogeneity of the microsco!ic
images high. &herefore/ the main advantage of the 2,%5 measured by fluorescent
s!ectrometry is that the data are obtained for the bul+ of the sam!le and do not de!end on the
!resence of aggregates or !articles in the sam!le I"J.
,n our case reactor 1/ with the higher '(D)&1* ratio/ has the lower amount of ($s because
of the higher amount of &1* in reactor 2.
&he !rocess denitrification with an effectiveness of 41: 7#18 and 80: 7#28 shows a
de!endenc which is caused on the fact that maybe in #2 there was more nitrate. *itrate is
converted into nitrogen gas in the denitrification ste! under ano-ic conditions.
,n our case the efficiency of denitrification is higher with an low '(D)&1* ration in variant
2. &his is unusual/ because normally increasing the ')* ratio increased the nitrate removal
rates 7nitrification8 I4J.
&he effectiveness of denitrification is low in both reactors and would need further ste!s of
denitrification/ usually in an anaerobic stage. ,n our case there were aerobic LLL conditions in
both reactors. &he usual denitrification needs anaerobic conditions. $ut there are some
bacteria which have the !ossibility for denitrification under aerobic coditions. 1im et al. have
analy6ed the aerobic denitrification of Hseudomonas !utida D.21 at different ')* ratios. &he
o!timal ')* ratio was 8 with a ma-imum nitrate removal rate of 254.9mg)l)h and a nitrate
removal efficiency of ;5.;:. &heir results suggest that H. !utida D.21 may be a good
candidate for aerobic wastewater treatment I4J.
nalytical Methods in $iological %ystems