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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