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  1. 1. Contributions of CINVESTAV to the study of bioreactors with simultaneous electron acceptors <ul><li>Removal of Trichlorophenol under partially-aerated methanogenic using a Fluidized Bed bioreactor </li></ul><ul><li>Removal of Perchloroethylene in partially-aerated methanogenic regime using a Fluidized Bed bioreactor </li></ul><ul><li>Removal of Perchloroethylene under M-D conditions </li></ul><ul><li>M-A and M-D bioreactors coupled to zero valent iron for PCE degradation </li></ul>
  2. 2. Removal of perchloroethylene in two methanogenic-denitrifying continuous systems Héctor M. Poggi-Varaldo CINVESTAV-IPN, Dept. Biotechnology and Bioengineering, Environmental Biotechnology R&D Group, México
  3. 3. Contents <ul><li>Acknowledgements </li></ul><ul><li>Abbreviations </li></ul><ul><li>Introduction </li></ul><ul><ul><li>Perchloroethylene </li></ul></ul><ul><ul><li>Simultaneous Electron Acceptor (SEA) systems </li></ul></ul><ul><li>Methodology </li></ul><ul><ul><li>Reactors set-up and operation </li></ul></ul><ul><ul><li>Methods </li></ul></ul><ul><li>Results and discussion </li></ul><ul><li>Conclusions </li></ul>
  4. 4. Acknowledgements <ul><li>Orgazing Committee and ENCB-IPN </li></ul><ul><li>CINVESTAV, CONACYT </li></ul><ul><li>Mr. Claudio Garibay-Orijel, </li></ul><ul><li>Mr. Rafael Hernández-Vera, Ms Paola Zárate-Segura </li></ul><ul><li>Prof. Elvira Ríos-Leal </li></ul><ul><li>Dr. Jaime García-Mena </li></ul>
  5. 5. Abbreviations <ul><li>B v organic loading rate per unit volume </li></ul><ul><li>CM complete mix reactor </li></ul><ul><li>DCE dichloroethylene </li></ul><ul><li>FBBR anaerobic fluidized bed biological reactor </li></ul><ul><li>I g biogas productivity </li></ul><ul><li>PCE perchloroethylene </li></ul><ul><li>TCE trichloroethylene </li></ul><ul><li>VC vinyl chloride </li></ul><ul><li> alpha factor </li></ul><ul><li> net increase </li></ul><ul><li> removal efficiency </li></ul><ul><li> loading ratio of organic matter as COD to nitrogen-nitrate </li></ul><ul><li> v PCE loading rate per unit volume </li></ul>
  6. 6. Introduction
  7. 7. <ul><li>Perchloroethylene </li></ul><ul><li>potentially hazardous </li></ul><ul><li>included in the priority list of hazardous pollutatns of USA and other countries (EPA, 1993) </li></ul><ul><li>widely used as a solvent in dry-cleaning industry as and a degreasing solvent in the metal and machinery industries for more than 50 years (HSIA, 1999) </li></ul><ul><li>in the 90’s, a world demand between 160 000 and 520 000 tonnes/yr has been recorded (EPA, 1993; WHO, 2000). </li></ul>
  8. 8. <ul><li>PCE is recalcitranr (not biodegradable) in aerobic conditions (Vogel and McCarty 1985) . Its biological transformation is generally carried out in anaerobic environments (van Eekert, 1999). </li></ul><ul><li>Previous works on biological treament and bioremediation of PCE have used anaerobic consortia that mediated the sequential reductive dehalogenation of PCE, presumably by cometabolism (Vogel y McCarty, 1985; Prakash y Gupta, 2000; Cope et al., 2001; López-Navarrete et al., 2003). </li></ul><ul><li>In most of these studies, accumulation of dichloroethylene (DCE) and vinyl chloride (VC) has been observed. A few works have been able to show the full transformation of PCE to ethene, after a long enrichment of anoxic consortia or using pure cultures of dehalorespiring bacteria (Mayor et al., 2002). </li></ul>
  9. 9. Series reactors and Simultaneous Electron Acceptor (SEA) systems
  10. 10. High chlorine content Penta-, tetra-, tri- Anaerobic Low chlorine content di-, and monochloro- Aerobic A good alternative: Series Reactors Brief example with a chlorinated organic compound Accumulation of
  11. 11. Series Reactors Anaerobic Reactor Reactor with second electron acceptor Tetrechloroethene TCE, DCE, VC Further removal of chlorinated aliphatics Garibay Orijel et al . (2005a) J. Chem. Technol. Biotechnol . In press Campos-Velarde et al . (1997). Battelle Press. What’s the problem with series reactors? 2 Reactors Costs X 2 Better try Simultaneous Electron Acceptors in One reactor Salto a la albóndiga
  12. 12. Protection via diffusion barrier in biofilm CH 4 CO 2 NO 3 - + org. matter N 2 +H 2 O Anaerobic zone Denitriying zone Liqued dif- fusion leayer Denitrifying microorganisms Methanogens Bulk liquid (adapted from López-Navarrete, 2002). Pollutant NO 3 - Concentration Carrier
  13. 13. <ul><li>= g COD/g 2nd electron acceptor in the influent </li></ul><ul><li>2nd electron acceptor: NO 3 - </li></ul><ul><li>lambda determines the percentage P of substrate that is channelled into methanogenesis or denitrification in SEA conditions </li></ul><ul><li>it can be demonstrated that P = 50% at  = 9 for M-D reactors </li></ul><ul><li>Garibay-Orijel et al. (2005b). J. Chem. Technol. Biotechnol . </li></ul>
  14. 14. Objectives
  15. 15. <ul><li>to evaluate and compare de performance of a fluidized bed bioreactor (FBBR) and complete mix reactors (CM) with suspended biomass, all of them fed with PCE as model chlorinated aliphatic and a low-moderate concentration of degradable organic matter as methanol </li></ul><ul><li>to assess the influence of the biochemical regime (full methanogenic versus M-D), and the effect of  in the M-D regime (18 and 9) on performance </li></ul>
  16. 16. Methodology
  17. 17. <ul><ul><li>Reactor setup: A Anaerobic Fluidized Bed Bioreactor (AFBBR); B Complete Mix Bioreactor (CM). 1A fluidized bed of bioparticles; 2A reservoir and feed of influent with a partial content of methanol; 2´A feed of stock of PCE in methanol; 3A recirculation; 4A liquid trap; 5A effluent reservoir; 6A biogas exit; 7A biogas sampling port; 8A activated carbon trap; 9A biogas measurement by brine meters; 1B suspended biomass; 2B reservoir and feed of influent with a partial content of methanol; 2´B feed of stock of PCE in methanol; 3B effluent reservoir; 4B biogas exit; 5B biogas sampling port; 6B activated carbon trap; 7B biogas sampling port. </li></ul></ul>
  18. 18. <ul><ul><li>Notes. </li></ul></ul><ul><ul><li>1000 mg COD-methanol/L in all periods; PCE: Perchloroethylene. a Anaerobic fluidized bed bioreactor at HRT= 1d, Vop= 2.8L, 35°C; b,c Complete mix reactor at HRT= 15 d, Vop= 2.5L, 35°C.; d Volumetric loading rate of organic matter in gCOD/(L.d), e Volumetric loading rate of PCE in mg PCE/(L.d); f Concentration of PCE in the influent, in mg/L; g Relation of volumetric loading and Nitrogen contend in Nitrate. </li></ul></ul>Operating conditions of bioreactors in the different periods of the experimental design Period 1 Period 2 Period 3    40
  19. 19. Operation and monitoring <ul><li>mesophilic conditions </li></ul><ul><li>glass column (2.8 L), loaded with 1 L of 1 mm granular activated carbon, colonized by an anaerobic consortium </li></ul><ul><li>methanol (1000mgCOD /L) </li></ul><ul><li>hydraulic residence time = 1 d (bed basis) </li></ul><ul><li>Response variables: </li></ul><ul><ul><li>Removal efficiency of organic matter (COD) </li></ul></ul><ul><ul><li>Removal efficiency of PCE </li></ul></ul><ul><ul><li>Concentration of less substituted chlorinated aliphatics </li></ul></ul><ul><ul><li>Specific methanogenic activity, specific denitrifying activity, specific oxgyen uptake rate </li></ul></ul>
  20. 20. T: transient period with 2vvd HRT= 1 d, TCP i = 80mg/L, Phe i = 20mg/L, COD i =1000 mg/L, mesophilic 15vvd <ul><ul><li>Dynamic performance of reactors </li></ul></ul><ul><ul><li>A: Removal efficiency of COD versus time; </li></ul></ul><ul><ul><li>B Biogas productivity; </li></ul></ul><ul><ul><li>C Increase of chloride anion. </li></ul></ul><ul><ul><li> FBBR </li></ul></ul><ul><ul><li>○ CM 1 </li></ul></ul><ul><ul><li>∆ CM 2 </li></ul></ul>M  = 18  = 9 Fluidized bed reactor Fluidized bed reactor Complete mix reactors Complete mix reactors Complete mix reactors  COD (%) I g (L biogas/L.d) ∆ Cl - (mg/L) Time (day)
  21. 21. Average performance of reactors 1/2
  22. 22. Average performance of reactors 2/2 Metabolites and dechlorination efficiencies
  23. 23. Poggi’s discrete divergence index n’ A = only green n’ B = only yellow n A = green plus white n B = yellow plus white Microbial community A Microbial community B Zárate-Segura et al. (2005). Battelle
  24. 24. Poggi’s discrete divergence index and dynamic divergence coefficient <ul><li> Poggi = (n’ A + n’ B )/(n A + n B ) </li></ul><ul><li>where </li></ul><ul><li>n’ A = number of bands in A that are not in B </li></ul><ul><li>n’ B = number of bands in B that are not in A </li></ul><ul><li>n A = total number of bands in A </li></ul><ul><li>n B = total number of bands in B </li></ul><ul><li>complete similarity 0   Poggi  1 complete divergence </li></ul><ul><li> Poggi = d(  Poggi )/dt; dynamic divergence coefficient </li></ul>. Zárate-Segura et al. (2005). Battelle
  25. 25. Lanes 1 and 2, methanogenic period with no PCE; Lanes 3 to 5, methanogenic period with 20 mg/L PCE; Lanes 6 to 8 methanogenic period with 40 mg/L PCE. All lanes contain 5  g of 16S rDNA PCR product. Poliacrylamide gel at 8% (Acrylamide/N-N´methylenbisacrylamide 37.5:1) Buffer TBE 1X, Urea -Formamide 10-50% (8M of Urea and 40%v/v formamide equivalent to 100% denaturing agents); 30 V, 13-15 mA, 8 h, 60°C. DGGE profiles of major bacterial communities present in fluidized bed bioreactor
  26. 26. Variation of bacterial communities in methanogenic FBBR and CM reactors in a period of operation with no PCE in the influent: Diagonal: comparison between start and end of the period for a given bioreactor Other cells: comparison between bioreactors
  27. 27. Variation of bacterial communities in methanogenic FBBR and CM reactors in a period of operation with 40 mg PCE/L for FBBR and CM2 and 20 mg PCE/L in CM1: Diagonal: comparison between start and end of the period for a given bioreactor Other cells: comparison between bioreactors
  28. 28. Variation of bacterial communities in methanogenic FBBR and CM reactors between periods with no PCE and with PCE in the feed: Diagonal: comparison between periods for a given bioreactor Other cells: empty
  29. 29. Ref.: 1. Juteau, P., Tremblay, D., Villemur, R., Bisaillon, J.-G. and Beaudet R. (2004). Analysis of the bacterial community inhabiting an aerobic thermophilic sequencing batch reactor (AT-SBR) treating swine waste. Appl. Microbiol. Biotechnol . 66, 115-122. 2. Ghosh, S. and LaPara, T.M. (2004). Removal of carbonaceous and nitrogenous pollutants from a synthetic wastewater using a membrane-coupled bioreactor. J. Industrial Microbiol. Biotechnol . 31, 353 –361. 3. Tartakovsky, B., Manuel, M.F., Beaumier, D., Greer, C.W. and Guiot, S.R. (2001). Enhanced selection of an anaerobic pentachlorophenol-degrading consortium. Biotechnol. Bioeng . 73, 476-483. Dynamic divergence indices of bacterial communities of several bioreactors
  30. 30. Conclusions
  31. 31. <ul><ul><li>- In Period 1 , methanogenic regime, AFBBR showed the best performance of the three reactors with higher values of both organic matter removal and PCE, in spite that volumetric loadings on AFBBR were 15-fold higher and some process stress would have been expected. </li></ul></ul><ul><ul><li>- During Period 2 , simultaneous M-D regime at  =18 gCOD/gN-NO 3 - , an improvement in performance of CM2 was observed. The other two reactors displayed similar performances than the corresponding in Period 1 . </li></ul></ul>
  32. 32. <ul><ul><li>-In Period 3 , simultaneous M-D regime at  = 9 gCOD/gN-NO 3 - , and concerning the two reactors fed with the highest PCE concentration 40 mg/L (FBBR and CM2), the FBBR outperformed CM2 in almost all the performance parameters and its metabolite profile was better than both of CM2 and CM1 (lower TCE, DCE, and VC in the effluent). </li></ul></ul><ul><ul><li>This pattern, along with highest dechlorination efficiency of FBBR strongly suggests that FBBR under M-D conditions may provide a more integral treatment to wastewaters polluted with significant concentrations of PCE. </li></ul></ul>
  33. 33. <ul><ul><li>The bacterial communities in the CMs were richer than those of FBBR during three operations periods </li></ul></ul><ul><ul><li>Generally, bacterial profiles in each reactor varied with time in a given period of operation. There was a relative higher stability of consortium in the FBBR as displayed by the lowest dynamic divergence coefficients values </li></ul></ul><ul><ul><li>The above described pattern was accompanied by a better biochemical peformance of FBBR (stable methanogenesis, high removal of PCE and lower concentrations of intermediate chlorinated aliphatics) </li></ul></ul>
  34. 34. <ul><ul><li>Despite relative variation of bacterial consortia with time, bioreactors showed steady state biochemical performance </li></ul></ul><ul><ul><li>PCE had a negative impact on the richness of CMs consortia whereas this impact was less noticeable in the FBBR. </li></ul></ul>
  35. 35. Questions and alibis [email_address] <ul><li>Me miro al espejo I look at myself in the mirror </li></ul><ul><li>y sólo veo but I can only see </li></ul><ul><li>la desnuda pared a mis espaldas. the bare wall behind me. </li></ul><ul><li>Me estremezco y me pregunto: I shiver and mutter: </li></ul><ul><li>¿quién soñará nuestros sueños? Who will dream our dreams? </li></ul><ul><li>¿quién peleará nuestras batallas? Who will fight our battles? </li></ul><ul><li>¿quién llorará nuestras derrotas? Who will cry for our defeats? </li></ul><ul><li>¿quién ganará nuestras victorias? Who will win our victories? </li></ul>Despedida Farewell