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Microbial DiversityMicrobial Diversityin a Dye Treating SBRin a Dye Treating SBR
bybyDr. Naeem ud dinDr. Naeem ud dinIslamia College Islamia College
PeshawarPeshawar
Biotreatment Biotreatment Different modes
A: using mixed culture
C: isolated enzymes
Dyes are hard to degrade, and often result in harmful Dyes are hard to degrade, and often result in harmful intermediatesintermediates
B: isolated organisms
1
6
7mg/L pH 7.
5
7
2
4
-
8
3
7 9
Figure 3.1 Schematic diagram of the experimental setup of the nitrifying 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.
A Specially Designed Airlift BR from a A Specially Designed Airlift BR from a previous Experiment for SND previous Experiment for SND achieving was used achieving was used
The Nitrogen Removing Process was The Nitrogen Removing Process was well established in that Reactorwell established in that Reactor
93 % of Ammonia and COD at an 93 % of Ammonia and COD at an HRT of 12 hrs.HRT of 12 hrs.
Table 1. Physical and Operational Conditions of the SBR
Parameter Value
Working volume (L)
Temperature (oC)
Dissolved oxygen (mg/l)
pH of bioreactor
Aeration: No aeration(minutes)
3.5
25 - 30
0.05 - 2.0
6.5-8.6
30: 120
The NITRIFYING MEDIUMThe NITRIFYING MEDIUM
Constituents QuantityNH4Cl (mg N L-1 ) 120
NaCl (mg L-1 ) 1000C6H12O6 (mg/L) 1000FeSO4 (mg L-1 ) 55.00
K2HPO4 (mg L-1 ) 140.00CaCO3(g L-1 ) 2.00
Trace metalsolution(ml/L)*
2
Yeast Extract(mg L-1 ) 10pH 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-MG dye-
textile industry, biological stain and textile industry, biological stain and antifungal. antifungal.
phytotoxic, a respiratory poison, and phytotoxic, a respiratory poison, and teratogenteratogen
This SBR was subjected to gradually This SBR was subjected to gradually increasing dye concentrationincreasing dye concentration
Optimization was achieved at a dye Optimization was achieved at a dye concentration of 25 mg/l and increased concentration of 25 mg/l and increased HRT of 36 hrsHRT of 36 hrs
In this experiment we used the activated In this experiment we used the activated sludge as a renewable biological resource sludge as a renewable biological resource to adsorb the usual environmental to adsorb the usual environmental concentrations of the MG dye. concentrations of the MG dye.
Synthetic DYE CONTAINING wastewater compositionSynthetic DYE CONTAINING wastewater composition
Constituents QuantityNH4Cl (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 metalsolution(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.
In that optimized state In that optimized state The Color and COD removal was 80 The Color and COD removal was 80
% % ammonia removal declined to 70 %. ammonia removal declined to 70 %. Biomass, 4 Biomass, 4 ++ 0.7 to 6 0.7 to 6 ++ 0.5 gm/l, SVI 0.5 gm/l, SVI
was in the range of 30 to 65 ml/gmwas in the range of 30 to 65 ml/gm
COD & Color removalCOD & Color removal
0
2
4
6
8
10
12
5 10 15 20 25 30 35 40 45 50 55 60
25
40
55
70
85
100
7 14 21 28 35 42 49 56 63 70 77 84
dye concentration(mg/L)
% R
emo
val
Time(day)
ColorCODpHPoly. (Color)Poly. (COD)
UV-Vis spectrophotometric scan of the biodecolorization of malachite green.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
700 661 621 581 541 501 461 421 381 341 301 261 221
Wave length(nm)
Ab
so
rba
nc
e
----0 hr ----2 hr ----4 hr ----6 hr
λmax 618 nm
Correlation between ammonia, biomass, dye Correlation between ammonia, biomass, dye concentration and OURconcentration and OUR
A B c
OUR
Knowledge about the microbial community Knowledge about the microbial community in a dye treating reactor would be useful in in a dye treating reactor would be useful in association with operational conditions, to association with operational conditions, to eliminate the pollutants efficiently. eliminate the pollutants efficiently.
likely to cause the domination of certain likely to cause the domination of certain groups of bacteriagroups of bacteria
This aspect inspired our interest This aspect inspired our interest to know the microbial community to know the microbial community evolved under the selective evolved under the selective pressure of the Dye in the SBR.pressure of the Dye in the SBR.
Microbial community structure in Microbial community structure in the Dye Treating SBR Sludgethe Dye Treating SBR Sludge
16S rRNA gene Library 16S rRNA gene Library Phylogenetic AnalysisPhylogenetic Analysis
PCR-amplification, clone library PCR-amplification, clone library construction and sequencingconstruction and sequencing
Bacterial universal primers Bacterial universal primers 27F (3′-AGAGTTTGATCATGGCTCAG-27F (3′-AGAGTTTGATCATGGCTCAG-
5′) and 5′) and 1492R (3′-1492R (3′-
TACGGYTACCTTGTTACGACTT-5′) TACGGYTACCTTGTTACGACTT-5′) were used for amplification.were used for amplification.
BLAST Analysis of the OTUs (culture- independent)
Taxon OTU* Clones/
Phylotype Closest Relative
Source in NCBI
Homology
HT-21 2 Caulobacter crescentus CB15 AE005673 97 % -Proteobacteria
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 %
-Proteobacteria
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 %
-Proteobacteria
HT-7 8 Hydrocarboniphaga effusa AY363245 95 %
HT-73 2 Bacteriovorax sp. AY294218 97 %
HT-13 2 Uncultured delta EF562566 99 %
-Proteobacteria
HT-67 1 Desulfovibrio carbinolicus DQ186201 98 %
Verrucomicrobia 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 % Unclassifiable
HT-30 1 Uncultured bacterium DQ413112 99 %
Phylogenetic analysisPhylogenetic analysis The obtained sequences were edited and The obtained sequences were edited and
aligned using the BioEdit software and aligned using the BioEdit software and CLUSTAL_W program (Thompson, 199724).CLUSTAL_W program (Thompson, 199724).
The sequences were compared to the known The sequences were compared to the known GenBank sequences using Basic Local GenBank sequences using Basic Local Alignment Search Tool (BLAST).Alignment Search Tool (BLAST).
Phylogenetic trees were constructed by Phylogenetic trees were constructed by neighbor-joining method with the MEGA neighbor-joining method with the MEGA package . Identical sequences were package . Identical sequences were recognized by phylogenetic tree analysisrecognized by phylogenetic tree analysis..
Phylo-genetic analysisPhylo-genetic analysis If the sequences similarity was more If the sequences similarity was more
than 97 %, they were considered as than 97 %, they were considered as identical and used for further identical and used for further phylogenetic analysis as an phylogenetic analysis as an operational taxonomic unit (OTU).operational taxonomic unit (OTU).
Culture-Independent Phylogenetic tree of the clones from the dye treating SBR,
HT-64*-27F
Uncultured beta DQ676335
HT-20*-27F
HT-96*-27F
Uncultured beta DQ837278
HT-69*-27F
Uncultured Thiobacillus AM167943
HT-43*-27F
Hydrogenophaga sp.DQ854968
HT-72*-27F
Burkholderia pseudomallei EU024169
HT-32*-27F
Ralstonia sp.AY509958
HT-47*-27F
Bacterium N57 EF207564
Beta Proteobacteria
HT-44*-27F
Stenotrophomonas maltophilia AB294557
HT-7*-27F
Hydrocarboniphaga effusa AY363245
HT-42*-27F
Pseudomonas fluorescens EF552157
HT-62*-27F
Acinetobacter haemolyticus AY586400
Gamma Proteobacteria
HT-93*-27F
Uncultured bacterium AY376698
HT-49*-27F
Uncultured bacterium EU192216
HT-16*-27F
Azospirillum rugosum AM419042
HT-21*-27F
Caulobacter crescentus AJ227757
HT-30*-27F
Uncultured bacterium DQ413112
Alpha Proteobacteria
HT-73*-27F
Bacteriovorax sp.AY294218
Desulfovibrio carbinolicus DQ186201
HT-67*-27F
Uncultured delta EF562566
HT-13*-27F
Delta Proteobacteria
HT-12*-27F
Uncultured eubacterium AF050559Verrucomicrobia
HT-66*-27F
Uncultured bacterium EF614090
0.02
Phylogenetic distribution profile of microbial community (Culture Independent) in the SBR.
Culture-Dependent MethodCulture-Dependent Method Nineteen isolates were selected from the SBR and Nineteen isolates were selected from the SBR and
their 16S rRNA genes were sequenced, and their 16S rRNA genes were sequenced, and compared with similar sequences of the reference compared with similar sequences of the reference organisms BLAST search. Figure 6 shows the organisms BLAST search. Figure 6 shows the phylogenetic tree based on the culture dependent phylogenetic tree based on the culture dependent isolates identified with sequences of the NCBI isolates identified with sequences of the NCBI BLAST. BLAST.
Some of the clones identified with the Some of the clones identified with the well-known well-known biodegraders, the notable being biodegraders, the notable being Dokdonella Dokdonella koreensis, Rhodobactor, Shingomonas koreensis, Rhodobactor, Shingomonas andand Paracoccus species.Paracoccus species.
Similarity of 16S rRNA gene sequences of the isolatesd l
Taxonomic Group Clone No.
Closest Relative Source in NCBI
Homology
AlphaproteobacteriaRhizobiales
Cdl2 Sinorhizobium sp. AM084032 99%
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%
Cdl6 Haematobacter missouriensis DQ342315 97%
Rhodobacterales
Sphingomonadales Cdl4Cdl11cdl14
Sphingomonas sp. DS4 EF494189 99 %
Cdl12 AF131297 99%
GammaproteobacteriaXanthomonadales
ml 23 Dokdonella koreensis strain NML 01-0233
EF589679 100 %
ActinobacteriaActinomycetales
25 Microbacterium hydrocarbonoxydans
AJ698726 98 %
Cdl 10Cdl 13
Uncultured bacteriumclone LR A2-35
DQ988316 100 %
ml 22 Uncultured bacterium clone SLB728
DQ787731 97 %
Unclassifiable
cdl 8 Uncultured bacterium clone aab65g10
DQ814239 99 %
Cdl 9 Uncultured bacterium clone WBB38
EU184871 100%
cd14 Estrogen-degrading bacterium DQ066439 99%
Sphingomonas taejonensis
Is No
Phylogenetic tree of isolates from the dye treating SBR
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 distribution, as illustrated by isolates in the SBR involved in the biotreatment MG.
Rhizobiales16%
Rhodobacterales21%
Sphingomonadales
21%
Xanthomonadales5%
Unclassifiable32%
Actinomycetales5%
All these groups well represented in the polluted environments
Table 2 shows the phylogenic affiliation and abundance of the Table 2 shows the phylogenic affiliation and abundance of the clones. The sequences identifying with 5 divisions of clones. The sequences identifying with 5 divisions of ProteoabacteriaProteoabacteria i.e i.e ά-, β-, γ- §-proteobacteriaά-, β-, γ- §-proteobacteria and and VerrucomicrobiaVerrucomicrobia groups were obtained. The groups were obtained. The β-, and γ-proteobacteriaβ-, and γ-proteobacteria were in high were in high abundance, valuing 24 % and 45 % of the total clones. The other abundance, valuing 24 % and 45 % of the total clones. The other small groups, consisting of small groups, consisting of ά-,§-proteobacteria ά-,§-proteobacteria andand Verrumicrobia Verrumicrobia groups, were 4 %, 9 % , and 2 % respectively. A moderate amount groups, were 4 %, 9 % , and 2 % respectively. A moderate amount of clones, about 9 %, ranked with the uncultured bacterial strains of clones, about 9 %, ranked with the uncultured bacterial strains with sequenced data in the NCBI. with sequenced data in the NCBI.
The similarity of six culture independent clones(HT-69, HT-47, HT-The similarity of six culture independent clones(HT-69, HT-47, HT-51, HT-66, HT-38, HT-7, HT-72), to the known sequences in the 51, 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 GenBank was lower than 95%. Due to difficulty in translating 16S rRNA gene sequence similarity values into nomenclature, it is rRNA gene sequence similarity values into nomenclature, it is assumed that assumed that similarity values to the known sequences similarity values to the known sequences below 95% may be regarded as evidence of the discovery below 95% may be regarded as evidence of the discovery of novel species(3). Thus there is ample possibility of of novel species(3). Thus there is ample possibility of unidentified bacteria in the SBR used in the present study. unidentified bacteria in the SBR used in the present study.
inferencesinferences
Θ SBR with good SVI, effectively removed MG, COD and SBR with good SVI, effectively removed MG, COD and nitrogen up to 25 mg/L dye, above which a strong nitrogen up to 25 mg/L dye, above which a strong inhibition of these processes was observed. inhibition of these processes was observed.
Θ The autotrophic nitrifying bacteria were not detected The autotrophic nitrifying bacteria were not detected at high dye concentration, acting as bio-indicators for at high dye concentration, acting as bio-indicators for the MG toxicity. The ammonia removal pathway was, the MG toxicity. The ammonia removal pathway was, however, present, an indication of the however, present, an indication of the microbialmicrobial redundancyredundancy..
inferencesinferences
Θ Majority of the sequences identified with the Majority of the sequences identified with the β-β- and γ- and γ-ProteobacteriaProteobacteria. pollutant degrading bacteria, like . pollutant degrading bacteria, like rhodobacteralesrhodobacterales, , sphingomonadalessphingomonadales were in plenty.were in plenty.
Θ The first time that MG treated in a nitrifying BR, with The first time that MG treated in a nitrifying BR, with its inhibitory effects, and microbial community its inhibitory effects, and microbial community monitored.monitored.
Θ Both culture-dependent and Culture independent Both culture-dependent and Culture independent methods must be used to have a true picuture of methods must be used to have a true picuture of microbial diversity.microbial diversity.
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