Sadaf Haghiri PhD student at Environmental engineering faculty Middle East Technical University. Supervisor: Assoc. Prof. Dr.

Sadaf Haghiri PhD student at Environmental engineering faculty Middle East Technical University Supervisor: Assoc. Prof. Dr. Bülent İçgen October 2016

2 Outline Intoduction Motivation for this research and background Aim of this study Methods and materials Results so far

Discharg or reuse of municipal waste water Introduction 3

4 Sludge Treatment Raw sludge or non-stabilized sludge tends to acidify digestion and produces odor. Stabilization to stop natural fermentation by converting its organic materials to water and biogas.

Treatment of Sludge 5

Anaerobic digestion process Anaerobic digestion is a bacterial process that is carried out in the absence of oxygen. The sludge is fed into large tanks and held for a minimum of 12 days to allow the digestion process to perform the four stages necessary to digest the sludge. 6

7 Upgrading anaerobic digestion pre-treatment: Limitation in digester: Slow rate of hydrolytic step, which is leading to slow degradation of the organic matter and high retention time. Solution: Use of chemical disintegration method: ozonation pre-treatment.

Upgrading anaerobic digestion phase seperation: Limitation in digester: In a single-stage system the prevailing ph (7 8) favours the methanogenic archaea, leading to non-optimum growth conditions for acidifying hydrolytic bacteria. Solution: Separation of process phases, where reactor parameters such as ph can be optimized for each phase to suit requirements of the microorganisms. 8

Evaluation of enhancement methods 9 Limitation in digester: Difficulty in getting information about dynamic of microorganisms population during the digestion. Solution: Use of molecular technique in biotechnology

Fluorescence in situ Hybridization (FISH) 10

Microorganisms and Oligonucleotides probes used in this study Groups Target organisms Probe Sequence (5' 3') DSM-No. Acidogenic Acetogenic sulfur reducing bacteria Denitrifiers Methanogenic bacteria Acidobacteria HoAc1402 5'- CTT TCG TGA TGT GAC GGG -3' DSM-22465 Acidobacteria SS_HOL1400 5'- TTC GTG ATG TGA CGG GC -3' DSM-22743 Clostridium CLOST 5'- CAG GAG ATG TCA AGT CTA GG -3' DSM-10612 Actinobacteria Actino-221 5'- CGC AGG TCC ATC CCA GAC -3' DSM-20639 Flavobacterium CFB563 5'- GGA CCC TTT AAA CCC AAT -3' DSM-18451 Syntrophobacterales DSBAC355 5'- GCG CAA AAT TCC TCA CTG -3' DSM-10017 Thermacetogenium GTAG992 5'- CCAGGTCCGCAGAGATGTCA-3' DSM-26808 Syntrophobacter SYN835 5'-GCA GGA ATG AGT ACC CGC-3' DSM-2805 Tepidanaerobacter GTE1002 5'- TCCGTTTCCGGTCTCTACCA-3' DSM-21804 Acetobacteraceae Aceto3B 5'- CAA CAT CCA GCA CAC ATC GT -3' DSM-8909 Desulfovibrio sp. DSV687 5'- TAC GGA TTT CAC TCC T -3' DSM-2480 Desulfobacter sp. DSB129 5'- CAG GCT TGA AGG CAG ATT -3' DSM-17510 Desulfobulbus sp. DBB660 5'- GAA TTC CAC TTT CCC CTC TG -3' DSM-10215 Desulfosarcina variabilis DSC193 5'- AGG CCA CCC TTG ATC CAA -3' DSM-2060 Desulfococcus multivorans DCC209 5'- CCC AAA CGG TAG CTT CCT -3' DSM-2059 Desulfuromonas acetexigens SRB385Db 5'- CGG CGT TGC TGC GTC AGG -3' DSM-1397 Pseudomonas sp. Pae997 5'- TCT GGA AAG TTC TCA GCA -3' DSM-1110 Achromobacter ACH221 5'- CGC TCY AAT AGT GCA AGG TC -3' DSM-21681 Bacillus Bmy843 5'- CTT CAG CAC TCA GGT TCG -3' DSM-4337 Acetate-denitrifying cluster DEN124 5'- CGA CAT GGG CGC GTT CCG AT -3' DSM-14793 Acetate-denitrifying cluster DEN581 5'- TGT CTT ACT AAA CCG CCT GC -3' DSM-5691 Methanobacteriales MB311 5'- ACC TTG TCT CAG GTT CCA TCT CC -3' DSM-2257 Methanomicrobials MG1200 5'- CRG ATA ATT CGG GGC ATG CTG -3' DSM-1539 Methanosarcina MS821 5'- CGC CAT GCC TGA CAC CTA GCG AGC -3' DSM-2256 Methanosaeta MX825 5'-TCG CAC CGT GGC CGA CAC CTA GC-3 DSM-17206 Archaea Archaea ARC 915 5'-GTG CTC CCC CCG CCA ATT CCT-3' DSM-17251

Representative images of FISH analysis Results A1 A2 A1) Bacillus (DSM-4337) 35% formamide, stained with DAPI. A2) Bacillus (DSM-4337) 35% formamide, stained with FITC labeled probe. 12

Representative images of FISH analysis B1 B2 B1) syntrophobacter (DSM-10017) 30% formamide, stained with DAPI. B2) syntrophobacter (DSM-10017) 30% formamide, stained with FITC labeled probe. 13

Novelty and background: 14 Most researchers have focused on the optimization of gas production, removal of organic matter and optimization of process. however there is no detailed report on the topic of microbial population dynamics involved during use of ozone pre- treatment process in one-stage digetion Previous studies on two-stage digestion have focused on novel equipment testing and hydrogen production and increase in energy yields at a similar retention time for food wastes. The literature is very sparse in contrasting one and two-stage digestion. There is no investigation about use of chemical (ozonation) pre-treatment in phase seperated anaerobic digestions.

This study aims at 15 1. Monitoring the impact of ozone pre-treatments on the microbial population dynamics and biogas production (single-stage anaerobic digester) 2. determining appropriate ozone dose as pre-treatment (single-stage anaerobic digester) 3. Determined optimum ozone dose will be applied as pre-treatment, to two-stage anaerobic digester and The microbial population dynamics and biogas production will be observed. 4. Finally, the study will be usefull for comperative analysis of effect of ozone pretreatment in terms of microbial flora and biogas production in single and twostage anaerobic digesters.

Anaerobic sludge culture and feedstock Method Anaerobic sludge culture of the reactors will be collected from Return Activated Sludge of anaerobic digester line of the Tatlar Wastewater Treatment Plant, Ankara Wastes used to feed the reactors will be obtained from a VRM wastewater treatment plant (vacuum rotation membrane) located in Middle East Technical University, Ankara. 16

Feedstock Method The reactors will be fed semi-continuously and pre-treatment will be implemented in different doses of ozonation for each reactor OSC-Modular 4HC, WEDECO ITT INDUSTRIES (2007) ozone generator 17

The schematic flow diagram, first part of experiment (one-stage digestion) 18

The schematic flow diagram,second part of experiment (two-stage digestion) 19

Measurement on a regular basis: total solids (TS) Total suspended sulids (TSS) volatile suspended solids(vss) total gas volume (TG) chemical oxygen demand (COD) ph CH4 percentage 20

Results 21 What have been done so far 1. Ordering the pure culture of bacterias. 2. Ordering the specific pobes for bacterias 3. Optimization of fluorescence in situ hybridization for pure culture of targeted bacteria. 4. Conducting the one and two-stage reactors set ups...on going

Thank you. 22