Microbial Diversity
in a Dye Treating SBR
by
Dr. Naeem ud din
Islamia College Peshawar
Biotreatment Different modes
A: using
mixed culture
B: isolated
organisms
C: isolated
enzymes
Dyes are hard to degrade, and often result in harmful
intermediates
8
7
9
5
1
2
3
7
4
7
m
g
/
L
p
H
6
7
Schematic diagram of the experimental setup .of the nitrifying
-
Figure 3.1
bioreactor. (1), feed tank; (2), feed pump; (3), Air pump; (4), Air meter; (5),
oxidation tank; (6), Settler; (7), pH, DO, probes data Logger; (8),
Microprocessors for controlling the cycles; (9), stirrer.
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A Specially Designed Airlift BR from a
previous Experiment for SND
achieving was used
The Nitrogen Removing Process was
well established in that Reactor
93 % of Ammonia and COD at an
HRT of 12 hrs.
Table 1. Physical and Operational Conditions
of the SBR
Parameter
Value
Working volume (L)
3.5
Temperature (oC)
25 - 30
Dissolved oxygen (mg/l)
0.05 - 2.0
pH of bioreactor
6.5-8.6
Aeration: No aeration(minutes)
30: 120
The NITRIFYING MEDIUM
Constituents
NH4Cl (mg N L-1 )
NaCl (mg L-1 )
C6H12O6 (mg/L)
FeSO4 (mg L-1 )
K2HPO4 (mg L-1 )
CaCO3(g L-1 )
Trace metal
solution(ml/L)*
Yeast Extract(mg L-1 )
pH
Quantity
120
1000
1000
55.00
140.00
2.00
2
10
7.8
*g/l; MgSO4·7H2O: 5, FeCl2·4H2O: 6, COCl2: 0.88, H3BO3: 0.1,
ZnSO4·7H2O: 0.1, CuSO4: 0.05, NiSO4: 1, MnCl2: 5, (NH4)6MO7O24·4H2O,
0.64 and CaCl2·2H2O: 5.
MG dye-


textile industry, biological stain and
antifungal.
phytotoxic, a respiratory poison, and
teratogen



This SBR was subjected to gradually
increasing dye concentration
Optimization was achieved at a dye
concentration of 25 mg/l and
increased HRT of 36 hrs
In this experiment we used the
activated sludge as a renewable
biological resource to adsorb the
usual environmental concentrations
of the MG dye.
Synthetic DYE CONTAINING wastewater composition
Constituents
Quantity
NH4Cl (mg N L-1 )
120
NaCl (mg L-1 )
1000
C6H12O6 (mg/L)
1000
FeSO4 (mg L-1 )
55.00
K2HPO4 (mg L-1 )
140.00
CaCO3(g L-1 )
2.00
Trace metal
solution(ml/L)*
2
Yeast Extract(mg L-1 )
10
MG (mg L-1 )
25
pH
7.8
*g/l; MgSO4·7H2O: 5, FeCl2·4H2O: 6, COCl2: 0.88, H3BO3: 0.1, ZnSO4·7H2O: 0.1, CuSO4: 0.05,
NiSO4: 1, MnCl2: 5, (NH4)6MO7O24·4H2O, 0.64 and CaCl2·2H2O: 5.


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In that optimized state
The Color and COD removal was 80
%
ammonia removal declined to 70 %.
Biomass, 4 + 0.7 to 6 + 0.5 gm/l,
SVI was in the range of 30 to 65
ml/gm
COD & Color removal
dye concentration(mg/L)
% Removal
5
10 15 20 25 30 35 40 45 50 55 60
100
12
85
10
8
70
6
55
Color
COD
pH
Poly. (Color)
Poly. (COD)
40
4
2
0
25
7
14 21 28 35 42 49 56 63 70 77 84
Time(day)
0.9
0.8
λmax 618 nm
----0 hr
----2 hr
----4 hr
----6 hr
Absorbance
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
700
661
621
581
541
501
461
421
381
341 301
261
Wave length(nm)
UV-Vis spectrophotometric scan of the
biodecolorization of malachite green.
221
Correlation between ammonia, biomass, dye
concentration and OUR
OUR
A
B
c
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Knowledge about the microbial
community in a dye treating reactor
would be useful in association with
operational conditions, to eliminate
the pollutants efficiently.
likely to cause the domination of
certain groups of bacteria
This aspect inspired our
interest to know the microbial
community evolved under the
selective pressure of the Dye
in the SBR.
Microbial community structure in
the Dye Treating SBR Sludge
16S rRNA gene Library
 Phylogenetic Analysis

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PCR-amplification, clone library
construction and sequencing
Bacterial universal primers
27F (3′-AGAGTTTGATCATGGCTCAG5′) and
1492R (3′TACGGYTACCTTGTTACGACTT-5′)
were used for amplification.
BLAST Analysis of the OTUs (culture- independent)
Taxon
-Proteobacteria
-Proteobacteria
-Proteobacteria
 -Proteobacteria
Verrucomicrobia
OTU*
Clones/
Phylotype
Closest Relative
Source in
NCBI
Homology
HT-21
2
Caulobacter crescentus CB15
AE005673
97 %
HT-16
1
Azospirillum rugosum
AM419042
97 %
HT-96
2
U. beta proteobacterium
clone 56S_1B_81
DQ837278
97%
HT-64
2
Unc. Beta. Proteobac
DQ676335
99 %
HT-72
1
Burkholderia seudomallei
EU024169
91%
HT-20
1
Un. beta proteobacterium
clone LKC3_102B.28
EF121350
96
HT-69
1
Uncultured Thiobacillus
AM167943
95 %
HT-43
1
Hydrogenophaga sp.
DQ854968
99 %
HT-32
4
Ralstonia sp.
AY509958
100 %
HT-47
1
Bacterium N57
EF207564
92 %
HT 42
2
Pseudomonas fluorescens
strain P17
EF552157
98 %
HT-62
7
Acinetobacter haemolyticus
AY586400
99 %
HT-44
1
Stenotrophomonas
maltophilia
AB294557
99 %
HT-7
8
Hydrocarboniphaga effusa
AY363245
95 %
HT-73
2
Bacteriovorax sp.
AY294218
97 %
HT-13
2
Uncultured delta
EF562566
HT-67
1
Desulfovibrio carbinolicus
DQ186201
98 %
HT-12
1
Uncultured eubacterium
AF050559
94 %
HT-49
3
Uncultured bacterium
EU192216
100 %
HT-66
1
Uncultured bacterium
EF614090
97 %
HT-93
1
Uncultured bacterium
AY376698
100 %
HT-30
1
Uncultured bacterium
DQ413112
99 %
99 %
Unclassifiable
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Phylogenetic analysis
The obtained sequences were edited
and aligned using the BioEdit software and
CLUSTAL_W program (Thompson,
199724).
The sequences were compared to the
known GenBank sequences using Basic
Local Alignment Search Tool (BLAST).
Phylogenetic trees were constructed by
neighbor-joining method with the MEGA
package . Identical sequences were
recognized by phylogenetic tree analysis.


Phylo-genetic analysis
If the sequences similarity was more
than 97 %, they were considered as
identical and used for further
phylogenetic analysis as an
operational taxonomic unit (OTU).
HT-64*-27F
Uncultured beta DQ676335
HT-20*-27F
HT-96*-27F
Uncultured beta DQ837278
HT-69*-27F
Uncultured Thiobacillus AM167943
Beta Proteobacteria
HT-43*-27F
Hydrogenophaga sp.DQ854968
HT-72*-27F
Burkholderia pseudomallei EU024169
HT-32*-27F
Ralstonia sp.AY509958
HT-47*-27F
Bacterium N57 EF207564
HT-44*-27F
Stenotrophomonas maltophilia AB294557
HT-7*-27F
Hydrocarboniphaga effusa AY363245
HT-42*-27F
Pseudomonas fluorescens EF552157
Gamma Proteobacteria
HT-62*-27F
Acinetobacter haemolyticus AY586400
HT-93*-27F
Uncultured bacterium AY376698
HT-49*-27F
Uncultured bacterium EU192216
HT-16*-27F
Azospirillum rugosum AM419042
HT-21*-27F
Caulobacter crescentus AJ227757
Alpha Proteobacteria
HT-30*-27F
Uncultured bacterium DQ413112
HT-73*-27F
Bacteriovorax sp.AY294218
Desulfovibrio carbinolicus DQ186201
HT-67*-27F
Delta Proteobacteria
Uncultured delta EF562566
HT-13*-27F
HT-12*-27F
Uncultured eubacterium AF050559
HT-66*-27F
Uncultured bacterium EF614090
0.02
Verrucomicrobia
CultureIndependent
Phylogenetic tree
of the clones from
the dye treating
SBR,
Phylogenetic distribution profile of microbial
community (Culture Independent) in the SBR.



Culture-Dependent Method
Nineteen isolates were selected from the SBR and
their 16S rRNA genes were sequenced, and
compared with similar sequences of the reference
organisms BLAST search. Figure 6 shows the
phylogenetic tree based on the culture dependent
isolates identified with sequences of the NCBI
BLAST.
Some of the clones identified with the well-known
biodegraders, the notable being Dokdonella
koreensis, Rhodobactor, Shingomonas and
Paracoccus species.
Similarity of 16S rRNA gene sequences of the isolates
d
l
Taxonomic Group
Alphaproteobacteria
Rhizobiales
Rhodobacterales
Sphingomonadales
Gammaproteobacteria
Xanthomonadales
Clone
No.No
Is
Cdl2
Closest Relative
Homology
Sinorhizobium sp.
Source in
NCBI
AM084032
ml26
X.tagetidis
X99469
99 %
ml 28
Rhodopseudomonas palustris
AB017261
99 %
Cdl5
Catellibacterium nectariphilum
AB101543
99%
Cdl8
Phyllobacteriaceae bacterium
AM403241
99%
Cdl1
Rhodobacter sphaeroides
D16424.1
100%
99%
Cdl6
Haematobacter missouriensis
DQ342315
97%
Cdl4
Cdl11
cdl14
Cdl12
Sphingomonas sp. DS4
EF494189
99 %
Sphingomonas taejonensis
AF131297
99%
Dokdonella koreensis strain
NML 01-0233
EF589679
100 %
AJ698726
98 %
DQ988316
100 %
DQ787731
97 %
DQ814239
99 %
EU184871
100%
DQ066439
99%
ml 23
Actinobacteria
Actinomycetales
25
Unclassifiable
Cdl 10
Cdl 13
ml 22
cdl 8
Cdl 9
cd14
Microbacterium
hydrocarbonoxydans
Uncultured bacterium
clone LR A2-35
Uncultured bacterium clone
SLB728
Uncultured bacterium clone
aab65g10
Uncultured bacterium clone
WBB38
Estrogen-degrading bacterium
Paracoccus kawasakiensis AB041770
Catellibacterium nectariphilum AB101543
cd15
cdl10
Uncultured bacterium DQ988316
cdl13
Haematobacter missouriensis DQ342317
cdl1
Rhodobacter sphaeroides RCAIL106G
Uncultured alpha AJ871061
cdl6
cdl7
lm-26
X.tagetidis X99469
cdl9
Uncultured bacterium EU184871
Ochrobactrum sp.EF125188
cdl8
Uncultured bacterium DQ814239
cdl2
Sinorhizobium sp.AM084032
lm-28
Rhodopseudomona palustris AB017261
cdl12
Sphingomonas sp.AF131297
cdi 4
cdl11
cdl14
Sphingomonas sp. EF494189
lm-22
Uncultured bacterium DQ787731
lm-27
Alpha proteobacterium AM411928
lm-23
Dokdonella koreensis EF589679
lm-25
Microbac. hydrocarbonoxydans AJ698726.
0.02
The isolates Identified with
- & - Proteobacteria
Phylogenetic tree
of isolates from
the dye treating
SBR
Phylogenetic distribution, as illustrated by isolates in
the SBR involved in the biotreatment MG.
Rhizobiales
16%
Unclassifiable
32%
Rhodobacterales
21%
Actinomycetales
5%
Xanthomonadales
5%
Sphingomonadale
s
21%
All these groups well represented
in the polluted environments


Table 2 shows the phylogenic affiliation and abundance of the
clones. The sequences identifying with 5 divisions of
Proteoabacteria i.e ά-, β-, γ- §-proteobacteria and
Verrucomicrobia groups were obtained. The β-, and γproteobacteria were in high abundance, valuing 24 % and 45 % of
the total clones. The other small groups, consisting of ά-,§proteobacteria and Verrumicrobia groups, were 4 %, 9 % , and 2
% respectively. A moderate amount of clones, about 9 %, ranked
with the uncultured bacterial strains with sequenced data in the
NCBI.
The similarity of six culture independent clones(HT-69, HT-47, HT51, HT-66, HT-38, HT-7, HT-72), to the known sequences in the
GenBank was lower than 95%. Due to difficulty in translating 16S
rRNA gene sequence similarity values into nomenclature, it is
assumed that similarity values to the known sequences
below 95% may be regarded as evidence of the discovery
of novel species(3). Thus there is ample possibility of
unidentified bacteria in the SBR used in the present study.
inferences
Θ
Θ
SBR with good SVI, effectively removed MG, COD and
nitrogen up to 25 mg/L dye, above which a strong
inhibition of these processes was observed.
The autotrophic nitrifying bacteria were not detected
at high dye concentration, acting as bio-indicators for
the MG toxicity. The ammonia removal pathway was,
however, present, an indication of the microbial
redundancy.
inferences
Θ
Θ
Θ
Majority of the sequences identified with the β- and γProteobacteria. pollutant degrading bacteria, like
rhodobacterales, sphingomonadales were in plenty.
The first time that MG treated in a nitrifying BR, with
its inhibitory effects, and microbial community
monitored.
Both culture-dependent and Culture independent
methods must be used to have a true picuture of
microbial diversity.
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