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Microbial Diversity in a Dye Treating SBR by Dr. Naeem ud din Islamia College Peshawar.

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Presentation on theme: "Microbial Diversity in a Dye Treating SBR by Dr. Naeem ud din Islamia College Peshawar."— Presentation transcript:

1 Microbial Diversity in a Dye Treating SBR by Dr. Naeem ud din Islamia College Peshawar

2 Biotreatment Biotreatment Different modes A: using mixed culture C: isolated enzymes Dyes are hard to degrade, and often result in harmful intermediates B: isolated organisms

3

4 A Specially Designed Airlift BR from a previous Experiment for SND achieving was used A Specially Designed Airlift BR from a previous Experiment for SND achieving was used The Nitrogen Removing Process was well established in that Reactor The Nitrogen Removing Process was well established in that Reactor 93 % of Ammonia and COD at an HRT of 12 hrs. 93 % of Ammonia and COD at an HRT of 12 hrs.

5 Table 1. Physical and Operational Conditions of the SBR Parameter Value Working volume (L) Temperature ( o C) Dissolved oxygen (mg/l) pH of bioreactor Aeration: No aeration(minutes) : 120

6 The NITRIFYING MEDIUM ConstituentsQuantity NH 4 Cl (mg N L -1 )120 NaCl (mg L -1 )1000 C 6 H 12 O 6 (mg/L)1000 FeSO 4 (mg L -1 )55.00 K 2 HPO 4 (mg L -1 ) CaCO 3 (g L -1 )2.00 Trace metal solution(ml/L)* 2 Yeast Extract(mg L -1 )10 pH7.8 *g/l; MgSO 4 ·7H 2 O: 5, FeCl 2 ·4H 2 O: 6, COCl 2 : 0.88, H 3 BO 3 : 0.1, ZnSO 4 ·7H 2 O: 0.1, CuSO 4 : 0.05, NiSO 4 : 1, MnCl 2 : 5, (NH 4 ) 6 MO 7 O 24 ·4H 2 O, 0.64 and CaCl 2 ·2H 2 O: 5.

7 MG dye- textile industry, biological stain and antifungal. textile industry, biological stain and antifungal. phytotoxic, a respiratory poison, and teratogen phytotoxic, a respiratory poison, and teratogen

8 This SBR was subjected to gradually increasing dye concentration 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 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. In this experiment we used the activated sludge as a renewable biological resource to adsorb the usual environmental concentrations of the MG dye.

9 Synthetic DYE CONTAINING wastewater composition ConstituentsQuantity NH 4 Cl (mg N L -1 )120 NaCl (mg L -1 )1000 C 6 H 12 O 6 (mg/L)1000 FeSO 4 (mg L -1 )55.00 K 2 HPO 4 (mg L -1 ) CaCO 3 (g L -1 )2.00 Trace metal solution(ml/L)* 2 Yeast Extract(mg L-1 )10 MG (mg L -1 )25 pH7.8 *g/l; MgSO 4 ·7H 2 O: 5, FeCl 2 ·4H 2 O: 6, COCl 2 : 0.88, H 3 BO 3 : 0.1, ZnSO 4 ·7H 2 O: 0.1, CuSO 4 : 0.05, NiSO 4 : 1, MnCl 2 : 5, (NH 4 ) 6 MO 7 O 24 ·4H 2 O, 0.64 and CaCl 2 ·2H 2 O: 5.

10 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, to gm/l, SVI was in the range of 30 to 65 ml/gm Biomass, to gm/l, SVI was in the range of 30 to 65 ml/gm

11 COD & Color removal

12 UV-Vis spectrophotometric scan of the biodecolorization of malachite green. λ max 618 nm

13 Correlation between ammonia, biomass, dye concentration and OUR A Bc OUR

14 Knowledge about the microbial community in a dye treating reactor would be useful in association with operational conditions, to eliminate the pollutants efficiently. 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 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. This aspect inspired our interest to know the microbial community evolved under the selective pressure of the Dye in the SBR.

15 Microbial community structure in the Dye Treating SBR Sludge 16S rRNA gene Library 16S rRNA gene Library Phylogenetic Analysis Phylogenetic Analysis

16 PCR-amplification, clone library construction and sequencing PCR-amplification, clone library construction and sequencing Bacterial universal primers Bacterial universal primers 27F (3′-AGAGTTTGATCATGGCTCAG- 5′) and 27F (3′-AGAGTTTGATCATGGCTCAG- 5′) and 1492R (3′- TACGGYTACCTTGTTACGACTT-5′) were used for amplification. 1492R (3′- TACGGYTACCTTGTTACGACTT-5′) were used for amplification.

17 BLAST Analysis of the OTUs (culture- independent)

18 Phylogenetic analysis Phylogenetic analysis The obtained sequences were edited and aligned using the BioEdit software and CLUSTAL_W program (Thompson, ). The obtained sequences were edited and aligned using the BioEdit software and CLUSTAL_W program (Thompson, ). The sequences were compared to the known GenBank sequences using Basic Local Alignment Search Tool (BLAST). 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. Phylogenetic trees were constructed by neighbor-joining method with the MEGA package. Identical sequences were recognized by phylogenetic tree analysis.

19 Phylo-genetic 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). 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).

20 Culture- Independent Phylogenetic tree of the clones from the dye treating SBR,

21 Phylogenetic distribution profile of microbial community (Culture Independent) in the SBR.

22 Culture-Dependent Method 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. 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. Some of the clones identified with the well-known biodegraders, the notable being Dokdonella koreensis, Rhodobactor, Shingomonas and Paracoccus species.

23 Similarity of 16S rRNA gene sequences of the isolates Is No

24 Phylogenetic tree of isolates from the dye treating SBR The isolates Identified with   Proteobacteria

25 Phylogenetic distribution, as illustrated by isolates in the SBR involved in the biotreatment MG. All these groups well represented in the polluted environments

26 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 γ- proteobacteria 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. 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 γ- proteobacteria 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, HT- 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 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. 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 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.

27 inferences 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. Θ 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.

28 inferences Θ Majority of the sequences identified with the β- and γ- Proteobacteria. pollutant degrading bacteria, like rhodobacterales, sphingomonadales were in plenty. Θ The first time that MG treated in a nitrifying BR, with its inhibitory effects, and microbial community monitored. Θ Both culture-dependent and Culture independent methods must be used to have a true picuture of microbial diversity.

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