BNR Wastewater Benchmarking Study via DNA Sequencing

The purpose of this report is to uncover the unique BNR wastewater (biological nutrient removal) characteristics of the FMBR and to confirm how they relate to the energy, biosolids, and footprint savings observed in the pilot demonstration.

In its recently completed Pilot Demonstration Project, JDL Global’s Facultative Membrane Bio-Reactor (FMBR) saved an estimated 77% in energy and 65% in biosolids disposal, as compared to the sequence batch reactor (SBR) that it replaced. The FMBR’s footprint was 75% smaller. The FMBR Pilot was meeting discharge permit limitations 30 days after the equipment was delivered to the site.

FMBR Pilot Demonstration Project

fmbr charts
digester benchmarking study

Phase I of DNA Analysis

In Phase I of this study, Microbe Detectives (MD) analyzed 13 samples of the FMBR Pilot, at the Plymouth, Massachusetts Municipal Airport, between May 2020 and May 2021. Standard MD 16S DNA sequencing methods were applied.

Phase II of DNA Analysis

In a 2nd phase project, MD 16S DNA sequencing data of FMBR Pilot samples was aggregated with MD 16S sequencing database of 675 samples from 18 municipal BNR wastewater processes, that are dispersed across New England, Midwest, Southwest, Rocky Mountains, and West Coast geographies in the USA. All data was anonymized. Statistical summaries of the presence and abundance of BNR wastewater microbes observed in each BNR wastewater process were compared, and are referred to as “BNR wastewater benchmarks.”

microbe detectives
BNR wastewater benchmarks

Carbon (C) Removal

Fermenting bacteria remove C by decomposing organic waste. They produce volatile fatty acids (VFAs) which are food for phosphate accumulating organisms (PAOs). PAOs remove phosphorus from wastewater biologically (BioP). The % abundance of Fermenting bacteria observed in FMBR Pilot samples averaged 4.2% vs 2.4% in BNR wastewater benchmarks. Tetrasphaera represented ~ 95% of fermenters. As a denitrifying PAO (DPAO), Tetrasphaera can utilize internally stored carbon both for phosphorus uptake and denitrification at the anoxic stage, resulting in increased phosphorus and nitrogen removal. In addition, DPAOs have stronger endogenous respiration which reduces sludge production [1].

fermenting bacteria BNR wastewater benchmarking study

Nitrogen (N) Removal

Simultaneous nitrification/denitrification (SND) bacteria have the potential to simultaneously complete nitrification and denitrification in one process step or reactor. This is a unique process which is usually operated at low dissolved oxygen concentrations (<0.5 mg/L) which results in aerobic conditions in the outside of the floc but anoxic conditions inside, allowing these bacteria to both oxidize ammonia aerobically and perform nitrate reduction in anoxic conditions. SND bacteria require 20-30% less oxygen and 40% less carbon than traditional N bacteria, and have stronger endogenous respiration which reduces sludge production [1]. The Plymouth FMBR Pilot operating data demonstrates obvious, efficient removal of nitrogen in a low DO environment <0.5 mg/L, and simultaneously in a single vessel. Over the 12 month Pilot Demo during 2020, total Nitrogen was reduced from an average 62.7 mg TN/L in the influent to 4.1 mg TN/L in the effluent.

The average abundance of N bacteria observed in FMBR Pilot samples was 17.6% vs 6.3% in BNR wastewater benchmarks. 94% of N bacteria observed in FMBR samples are known to have the potential of performing simultaneous nitrification/denitrification. Dechloromonas (avg. 8.3% in FMBR vs 1.0% in BNR wastewater benchmarks), Pseudomonas (avg. 8.1% in FMBR vs 3.1% in BNR wastewater benchmarks), and Tetrasphaera (avg. 4.0% in FMBR vs 2.4% in BNR wastewater benchmarks), were detected in all 13 FMBR samples, often at a relatively high % abundance. In other studies, Dechloromonas, a known phosphate-accumulating organism (PAO), has been observed in high abundance in alternating anoxic and aerobic conditions, enabling nitrification and denitrification [2, 3]. Pseudomonas has been observed to be capable of performing heterotrophic nitrification and then denitrify their nitrification products under aerobic conditions [4]. These observations combined with an absence of traditional N removal bacteria observed in FMBR Pilot samples, suggests SND bacteria may play a key role in N removal.
SND BNR wastewater benchmarking study

Phosphorus (P) Removal

Phosphorus accumulating organisms (PAOs) have a unique metabolic ability to take up and store volatile fatty acids (VFAs) such as acetic acid during anaerobic conditions. PAOs consume polyphosphate for energy during anaerobic conditions and take up organics and store them as polyhydroxy acetate (PHA). They then consume PHA for energy during aerobic conditions and take up phosphate to store energy as a polyphosphate granule.

PAOs were observed at an average 12.4% in FMBR Pilot samples vs 4.1% in BNR wastewater benchmarks. Tetrasphaera has been shown to be a very robust PAO and was identified in all 13 samples. In the first three samples it was observed in the range of 14%-16%. When Tetrasphaera % abundance decreased, Dechloromonas % abundance increased to similar high levels. Dechloromonas was observed in the range of 12 to 18% relative abundance in four samples. Rhodocyclus was detected at low levels in all 13 samples. Overall PAO % abundance in FMBR samples was the highest among all benchmarks.

PAO BNR wastewater benchmarking study

DNA Data Supports and Helps Explain Operational Performance

The Plymouth Airport FMBR results demonstrate obvious, efficient, simultaneous removal of C, N, and P in a low DO environment <0.5 mg/L, in a single vessel. Based on average daily values, BOD was reduced from 371 mg/L to non-detect. TSS was reduced from 79 mg/L to non-detect. Total Nitrogen was reduced from 62.7 mgTN/L to 4.1 mgTN/L. Phosphorus was reduced 10.0 mgP/L to <1.0 mgP/L. These results were reliably below permit discharge limits of BOD<30, TN<10 and TSS<30, (mg/L). Energy was reduced 77% compared to the legacy SBR WWTP. The volume of residual biosolids requiring offsite disposal was reduced 65%. The footprint of the WWTP was reduced 75%.

BNR wastewater benchmarking study
Microbial data generated by next generation DNA sequencing supports and helps explain the operational performance observed.

  • The % abundance of Fermenting bacteria observed in FMBR Pilot samples averaged 1.75 times more than BNR wastewater benchmarks. Fermenting bacteria remove C by decomposing organic waste. They produce volatile fatty acids (VFAs) which are food for phosphate accumulating organisms (PAOs). This higher than normal abundance of fermenting bacteria observed in FMBR samples correlates well with the observed reduction of residual biosolids.
  • The average abundance of N removal bacteria observed in FMBR Pilot samples was 2.8 times more than BNR wastewater benchmarks. 94% of N bacteria observed in FMBR samples are known to have the potential of performing simultaneous nitrification/denitrification. This higher than normal abundance of N bacteria, and specifically SND bacteria, observed in FMBR samples correlates well with the observed energy savings, and low DO (<0.5 mg/L) condition.
  • The average abundance of P removal bacteria observed in FMBR samples was 3.0 times more than BNR wastewater benchmarks. This supports the claim of simultaneous removal of P and N in the same vessel. The higher than normal P removal bacteria also supports the claim that a higher level of VFAs are generated than normal, due to a greater decomposition of organic matter. Since VFAs are known to serve as the key food for P removal bacteria (PAOs), it makes sense that PAOs are higher than normal.
  • Key microbes common in traditional BNR wastewater processes were largely absent in FMBR samples. This included Ammonia Oxidizing Bacteria (AOBs) (avg. 0.1% in FMBR samples), Nitrite Oxidizing Bacteria (NOBs) (avg. 1.0%), and Nitrate Reducing Bacteria (NRBs) (avg. 0.1%). This is what we would expect to observe in a low DO SND condition, such as with the FMBR.

In the end, the DNA and operational data confirmed the results – simultaneous removal of C, N, and P, in a single tank, with a surprisingly small amount of energy, footprint, and biosolids waste.

Principal Author

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John Tillotson, MSCE, is the Managing Partner at Microbe Detectives (MD) and its environmental consulting practice WaterTrust. Over the past 5 years, John has been developing MD’s DNA sequencing services with a specialization in water reclamation, biological nutrient removal (BNR wastewater), and anaerobic digestion in municipal and industrial systems. Prior to MD, he had over 25 years experience in water/ environment, data, and IoT. He holds an M.S. in Civil Engineering from Tufts University, a B.S. in Geochemistry from West Chester University, and a Toxics Use Reduction Planner Certification from the Massachusetts Toxics Use Reduction Institute.

Contributors and Science Advisors

alison ling

Alison Ling, Ph.D., P.E., is an Environmental Engineer with Barr Engineering Company in Minneapolis, MN, and Scientific Advisor at Microbe Detectives. At Barr, she is engaged in front-end design and troubleshooting for water and wastewater treatment projects. Her work includes process modeling, bench testing, and technical advising for clients in the power, mining, food and beverage, and municipal sectors. Ali holds a PhD in Civil Engineering from the University of Colorado at Boulder, where she split her time between Environmental Engineering and Microbial Ecology lab groups.

rahman

Arifur Rahman, Ph.D., P.E., is a Wastewater Process Specialist at Jacobs Engineering Group Inc., in Dallas, TX. His technical expertise is on carbon diversion and recovery from wastewater, energy recovery, BNR wastewater, process technology design, and wet weather flow management. He is actively involved in various wastewater research and development related projects. He holds a MS and Ph.D. degree from The George Washington University and a B.Sc degree in Civil Engineering from Bangladesh University of Engineering and Technology. He is a licensed professional engineer in the State of Texas.

References

[1] Kristiansen, R., Nguyen, H., Saunders, A. et al. “A metabolic model for members of the genus Tetrasphaera involved in enhanced biological phosphorus removal.” ISME J 7, 543–554 (2013).

[2] Xu, D., Liu, S., Chen, Q. et al. “Microbial community compositions in different functional zones of Carrousel oxidation ditch system for domestic wastewater treatment.” AMB Expr 7, 40 (2017). https://doi.org/10.1186/s13568-017-0336-y

[3] Lifang Luo, Junqin Yao*, Weiguo Liu et al. “Comparison of bacterial communities and antibiotic resistance genes in oxidation ditches and membrane bioreactors.” Nature Portfolio, Scientific Reports (2021)

[4] Jin, R., Liu, T., Liu, G. et al. “Simultaneous Heterotrophic Nitrification and Aerobic Denitrification by the Marine Origin Bacterium Pseudomonas sp. ADN-42.” Appl Biochem Biotechnol 175, 2000–2011 (2015).

[5] Bergey’s Manual of Systematic Bacteriology, Edition Eight, by R.E. Buchanan and N.E. Gibbens, page 517.

[6] Strous M.; et al. “Deciphering the evolution and metabolism of an anammox bacterium from a community genome.” Nature. 440 (7085): 790–794. (2006).

[7] Lv, Xiao-Mei et al. “A comparative study of the bacterial community in denitrifying and traditional enhanced biological phosphorus removal processes.” Microbes and environments vol. 29(3): 261-268. (2014).