Enhanced Biological Phosphorus Removal Using Crude Glycerol (from biodiesel production) in lieu of VFA Supplementation

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2 Enhanced Biological Phosphorus Removal Using Crude Glycerol (from biodiesel production) in lieu of VFA Supplementation PNCWA October 23, 2012 Boise, Idaho Erik R. Coats, PE, Ph.D. University of Idaho Zach Dobroth, EIT Brown and Caldwell

3 Presentation Outline Background Phosphorus and the water environment Enhanced Biological Phosphorus Removal (EBPR) Crude Glycerol Research Goals Experimental Setup Results Conclusions Practical Impacts

4 Phosphorus Issues Phosphorus (P) in the water environment stimulates excess growth of algae and microorganisms; can lead to advanced eutrophication Increasingly stringent effluent P regulations for WWTPs Boise River limits approaching 0.07 mg/l Spokane River ~ 0.10 mg/l P Species Ortho-P (reactive phosphate) Poly-P (non-reactive phosphate) Organic P

5 Enhanced Biological P Removal (EBPR) Natural, biological process to remove P from wastewater Principally targets Ortho-P removal Requires cycling activated sludge repetitively through anaerobic and aerobic environments EBPR theory prescribes volatile fatty acids (VFAs) Principally acetic and propionic acid P is removed through sludge wasting

6 Typical EBPR Process Configuration

7 EBPR Bacteria Enrich for Phosphorus Accumulating Organisms (PAOs) Uptake and store excess P as polyphosphate granules Glycogen Accumulating Organisms (GAOs) are considered undesirable Theoretically compete for carbon substrate without removing P

8 PAO Metabolisms Anaerobic: Store carbon (model substrate = acetate) as PHA Hydrolyze polyp for energy; release P into bulk solution Glycogen provides carbon, electrons, and additional energy Aerobic: Use PHA for growth and to replenish glycogen; uptake excess P, store as polyp

9 Typical EBPR C, P Cycling Patterns

10 Alternate Organic Carbon Sources? Consider free industrial waste streams? Crude glycerol (CG) byproduct of biodiesel production Every 12.6 L biodiesel = 1.12 L CG Crude Glycerol Glycerol: 50%+ by volume Alcohol (typically methanol) Residual catalyst (salt) Free fatty acids CH 2 - OOC- R 1 CH- OOC- R 2 + 3R OH Catalyst Glycerol metabolized as sugar All CG carbon forms are direct precursors for PHA synthesis Use CG to stimulate EBPR? Biodiesel R 1 -COO- R R 1 -COO- R + CH 2 -OH CH- OH CH 2 - OOC- R 3 R 1 -COO- R CH 2 -OH Triglyceride Alcohol Fa;y Acid Esters Glycerol

11 Research Goal To examine the suitability of crude glycerol as a direct substitute for traditional EBPR substrate in a process utilizing a mixed microbial population from a full-scale WWTP. Investigations conducted over a 723 day period.

12 Experimental Setup Sequencing Batch Reactors (SBRs) Inocula, wastewater from Moscow, ID, WWTP VFA-rich wastewater from lab-scale primary solids fermenter CG from pilot-scale biodiesel facility, University of Idaho All reactors: Volume = 2 L, SRT = 10 days, HRT = 18 hr Influent 1 hr 4.25 hr Effluent Feed Anaerobic Aerobic Settle Decant Time One Cycle = 6 hours

13 Reactor Descriptions Reactor R-EBPR V-EBPR G-EBPR V2-EBPR G2-EBPR Substrate (% by volume) 100% raw wastewater 90% raw wastewater, 10% fermenter liquor Raw wastewater with 0.2 ml CG/L Initially: 90% raw wastewater, 10% fermenter liquor After day 385: Raw wastewater with 0.2 ml CG/L Initially: Raw wastewater with 0.2 ml CG/L After day 385: 90% raw wastewater, 10% fermenter liquor We will be referencing these Reactor names throughout the presentation

14 Results and Discussion Part 1 EBPR Performance: VFA vs. CG Augmentation Phosphorus Removal Carbon Utilization Effects of Substrate on Process Resiliency and Stability A Permanent Change in Substrate A One-time Switch in Substrate Carbon Substrate Effects on Anaerobic EBPR Metabolisms P:C Ratio Anaerobic Energetics PAO and GAO Quantification

15 Phosphorus Cycling, VFA-fed Effluent ortho-p = 0.09 mg/l ±0.08 mg/l

16 Phosphorus Cycling, CG-fed Effluent ortho-p = 0.06 mg/l ±0.04 mg/l

17 Phosphorus Cycling, Raw WW-fed

18 Carbon Cycling, VFA-fed

19 Carbon Cycling, CG-fed

20 VFA vs. CG Performance Analysis Anaerobic P release much higher in VFA-fed reactors than CG-fed and raw WW-fed reactors Carbon cycling consistent with EBPR theory CG-fed population exhibited more glycogen cycling Excellent P removal in both VFA-fed and CG-fed reactors; not in raw WW-fed reactors Of note: on 8 occasions the CG-fed bacteria exhibited zero anaerobic P release Fully inconsistent with current EBPR theory

22 Step 1 - Changing Substrate Permanently How will bacteria perform P removal when substrate is permanently switched? Reactors V2- and G2-EBPR established using inocula from their respective parents.initially operated receiving the normal substrate (i.e., V2-EBPR receiving VFA-rich substrate and vice versa) for a time period greater than three SRTs. V2-EBPR V-EBPR G-EBPR Transfer bacteria to inoculate new reactor V2-EBPR G2-EBPR Switch the substrate permanently receiving crude glycerol G2-EBPR receiving VFArich wastewater

23 Acclimating to the New Substrate VFA-enriched bacteria maintained continuous process stability Despite receiving both fewer VFAs and a more diverse organic carbon substrate In contrast, the CG-enriched bacteria experienced significant process upset Despite receiving the prescribed VFA-rich substrate

24 Substrate Effects on Bacterial Population Comparing lanes 5, 6: Bacterial diversity increased with the switch to CG. Two new signals can be observed (A and B). Investigations are ongoing to identify the phylogeny of these signals. Comparing lanes 1, 2: Substrate switch decreased the microbial diversity in this reactor, which could explain the process instability following the substrate switch. Dominant microbial populations appeared substrate independent. At least one dominant signal unique to the successful EBPR MMC that was not present in the R-EBPR reactors (signal C).

25 Step 2 - A Temporary Shift in Substrate Full-scale EBPR treatment plants can experience reduced concentrations of organic carbon through. Fermenter failure or maintenance Infiltration and Inflow Can CG be used to maintain process stability? Our permanent substrate switch investigations suggest yes

26 Switching from VFAs to CG

27 Switching from CG to VFAs

29 The P:C Ratio In accordance with EBPR theory. polyp n polyp n-1 + ATP (energy) + P (into solution) Anaerobic P release indicates that a cascade of biochemical reactions are induced which will lead to successful, stable EBPR P release is theoretically exclusive to PAOs P:C is a useful metric/indicator in process trouble-shooting The C represents VFAs that the bacteria consume anaerobically Direct relationship between the P:C ratio and ph. P:C = 0.16 * AN ph 0.55 Equation intrinsically linked to energy requirement to process VFAs

30 P:C Results Theory vs. Empirical VFA-fed bacteria Theory: Actual: (average of 0.27) P:C decreased as ph increased; inconsistent with theory CG-fed bacteria Theory: Actual: (average of 0.16) No observed link between ph and P:C

31 P:C Results What can we learn? P:C for real WW-based operations will vary starkly from theory Should be < theory due to presence of non-paos Activated sludge contains non-pao bacteria that consume VFAs Considering its theoretical basis (activated sludge HIGHLY enriched in PAOs), the P:C ratio is biased toward unrealistic environments For P:C = 0 PolyP energy not needed Energy generated via other mechanisms

32 Beyond P:C -- Anaerobic Energetics Energy (as ATP) is required anaerobically for VFA PHA Bacteria can generate energy (as ATP) through. polyp hydrolysis Glycogen degradation For the CG reactors, glycerol degradation Did the CG-fed bacteria generate enough energy for successful EBPR without polyp hydrolysis? P:C analysis did not/cannot answer this question We developed a quantitative assessment of the anaerobic energetics

33 Assessing CG, VFA-fed Anaerobic Energetics From the table below. Energy balance for the CG-fed bacteria, not the VFA-fed bacteria The addition of glycerol provided sufficient energy that little polyp was required Considering the VFA-fed bacterial results, it would appear that PAOs maintain other anaerobic ATP production mechanisms Some evidence of ATP synthesis associated with the ATP Synthase mechanism (normally considered an aerobic process) ATP Required (mmol) VFA Uptake & Transport Averages ATP Produced (mmol) VFA Activation to CoA Total PolyP Glycogen Glycerol Total % ATP accounted for CG- fed MMC % VFA- fed MMC %

35 PAOs vs. GAOs PAOs: Model EBPR bacterium Capable of performing all the theoretical metabolisms GAOs: Considered competitors to PAOs..therefore theoretically BAD Can use VFAs and Glycogen just like PAOs.but do not exhibit polyp cycling and thus no anaerobic P release Supposedly not capable of excess P uptake We applied quantitative PCR to quantify PAOs and GAOs PCR primers were used that specifically target the 16S rdna of putative PAOs and GAOs

36 Are GAOs actually capable of EBPR?? PAOs GAOs GB lineage Operational Reactor Day R- EBPR V- EBPR G- EBPR V2- EBPR G2- EBPR ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± None detected 457 None detected 485 None detected 520 None detected 644 None 0.06± ± ± ±0.02 detected 716 None 4.90± ± detected 385 None detected 457 None detected 485 None detected 520 None detected 644 None 0.07± ± ± ±0.01 detected 716 None 3.41± ± detected

37 Conclusions Crude glycerol: a useful source of organic carbon for EBPR As a permanent replacement to VFAs CG-enriched activated sludge may not be as resilient As a supplement during low influent VFA periods Contradicting current EBPR theory, polyp hydrolysis for energy is not absolutely necessary for process success So-called GAOs may not be so detrimental to EBPR as other research would suggest

38 Practical Impacts & Future Research Extrapolating from our results 10 mgd WWTP would require 2,000 gallons per day of CG This quantity of CG is generated from a 2 MG per year full-scale biodiesel plant (a small biodiesel production facility) Ongoing research Leverage this substrate and the unexpected metabolic responses to further investigate EBPR metabolisms Investigate if GAOs = PAOs

39 Thank you!

40 TEMPLATE

41 General Metabolic Observations CG can be used to achieve excellent P removal in lieu of traditional EBPR substrate. However. The prototypical anaerobic EBPR metabolisms were not consistently induced (specifically zero anaerobic P release) The CG-fed MMC also readily consumed the non-vfa carbon substrate anaerobically. Why or how does EBPR function successfully without the model substrate and without anaerobic P release? Interrogation of the anaerobic process stoichiometry and energetics revealed some insights on these unexpected metabolic observations

42 quantitative PCR (qpcr) Extract DNA from the activated sludge Copy a DNA fragment from the target bacteria (i.e., PAOs, GAOs) Target a DNA fragment associated with general protein synthesis Highly conserved DNA sequence across the target bacteria The DNA copying process occurs multiple times, generating exponential increase in quantities of the target DNA PCR is bias toward dominant microbial populations Because of exponential copying, more initial target bacteria = more ultimate DNA As the DNA copying process occurs, each new piece of DNA binds with a fluorescent marker Measure fluorescence..corresponds with actual quantity of the target bacteria

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