DNA sequencing for the removal of biological nutrients


With most biological nutrient removal (BNR) processes from wastewater, the identities and abundances of microbes are not directly measured or tracked because BNR microbes are very small and not identifiable with standard testing from the water. ‘industry. With modern molecular testing methods, such as next-generation DNA sequencing, it is now possible to digitize the ecology of BNR processes at an affordable price, identifying, quantifying, tracking and bio-cartoning nearly all microbes present.

BNR DNA Sequencing Case Study

The Optional Membrane Bioreactor (FMBR) is a new type of membrane bioreactor (MBR) wastewater treatment technology that has recently been documented to reduce energy costs by 77% compared to a sequential batch reactor (SBR) that ‘he replaced in a demonstration pilot. It also saved 65% on biosolids disposal and had a 75% smaller footprint.

FMBR technology was invented by Jiangxi JDL Environmental Protection Co., Ltd. (JDL) of Nanchang, China in 2008. JDL claims 47 invention patents in the United States, United Kingdom, France, Japan, China and other countries, and more than 3,000 systems installed and commissioned in 19 countries.

FMBR is a biological process optimized to simultaneously remove carbon, nitrogen and phosphorus from wastewater in one step, at low energy. Its design is based on proprietary environmental control strategies that select specific types of BNR microbes without the need to add any inputs other than a continuous supply of sewage and a small amount of electrical energy to power the system. The outlets are clean water effluents that meet NPDES permit requirements or are reused on site for water conservation and sustainability benefits. The design features make it an efficient decentralized wastewater treatment system (DEWATS).


FMBR – How is it different?

Some modern MBR systems complete nitrification and denitrification simultaneously, under low dissolved oxygen (DO) conditions, saving energy and footprint. Normally, phosphorus is removed in a different process. With FMBR, phosphorus is biologically removed in the same reactor and the same ecological environment where nitrification and denitrification occur simultaneously, further reducing the footprint and costs.

FMBR pilot DNA sequencing to identify which bacteria are there and how many?

In Phase I of this study, Microbe Detectives (MD) analyzed 13 samples from the FMBR pilot, between May 2020 and May 2021. Standard MD 16S DNA sequencing methods were applied, identifying almost all bacteria and archaea at the genus level or higher and their relative percentage of abundance.

Data analysis was specialized for BNR applications in order to create key observations. The main BNR microbes of interest that have been shown to perform beneficial biological nutrient removal functions were identified and measured for their relevant percentage abundance, summarized in Table 1.

How do BNR’s biocharacteristics compare?

The MD 16S DNA sequencing data from the FMBR pilot samples was aggregated with the MD 16S sequencing database of 675 samples from 18 municipal wastewater BNR processes that are scattered across geographies of New Brunswick. England, Midwest, Southwest, Rockies, and West Coast of the United States, with average flow rates ranging from less than 1 million gallons per day (mgd) to over 100 mgd, summarized in the table 2. All data has been anonymized. Statistical summaries of the presence and abundance of BNR microbes observed in each BNR process (“benchmark”) were compared.

Key observations

Carbon removal (C). Fermentation bacteria remove C by breaking down organic waste. They produce volatile fatty acids (VFAs), which are food for phosphate accumulating organisms (OPAs). PAOs biologically remove phosphorus from wastewater (BioP). The percentage of abundance of fermenting bacteria observed in the FMBR pilot samples averaged 4.2% versus 2.4% in the BNR references. Tetrasphaera represented around 95% of the fermenters. As a denitrifying PAO (DPAO), Tetrasphaera can use internally stored carbon for both phosphorus uptake and anoxic stage denitrification, resulting in increased phosphorus and nitrogen removal. In addition, DPAOs have a stronger endogenous respiration, which reduces the production of sludge. [1].

Removal of nitrogen (N). The FMBR pilot system relied primarily on simultaneous nitrification and denitrification (SND) bacteria to remove nitrogen. the outside of the floc but anoxic conditions inside. This allows these bacteria to both oxidize ammonia aerobically and to effect nitrate reduction under 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.

nitrification denitrification

An average abundance of 17.6% of N bacteria was observed in FMBR samples vs 6.3% in BNR benchmarks, of which 94% were SND bacteria. Dechloromonas (mean 8.3% in FMBR vs. 1.0% in BNR benchmarks), Pseudomonas (mean 8.1% in FMBR vs. 3.1% in BNR benchmarks) and Tetrasphaera (mean 4.0% in FMBR vs. 2.4% in BNR markers), were detected in all 13 FMBR samples, often at a relatively high percentage abundance.

Elimination of phosphorus (P). PAOs have a unique metabolic capacity to absorb and store VFAs, such as acetic acid under anaerobic conditions. PAOs consume polyphosphate for energy under anaerobic conditions and absorb organics and store them as polyhydroxy acetate (PHA). They then consume PHA to produce energy under aerobic conditions and take up phosphate to store energy in the form of polyphosphate granules.

PAOs were observed at an average of 12.4% in the FMBR pilot samples versus 4.1% in the BNR references. Tetrasphaera was found 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% to 16%. The overall percentage of PAO abundance in the FMBR samples was the highest among all benchmarks.

Traditional BNR microbes are absent. Key microbes common in traditional BNR processes were largely absent. This included ammonia oxidizing bacteria (AOB) (mean 0.1% in FMBR samples), nitrite oxidizing bacteria (NOB) (mean 1.0%), and nitrate reducing bacteria (NRB) (mean 0.1%). Under conditions of low dissolved oxygen SND, these microbes should be absent because the environmental conditions are not suitable.

DNA data and operational data confirm the results. Daily tributary and effluent test data of C, Total Nitrogen (TN), Biological Oxygen Demand (BOD) and Total Suspended Solids (TSS) removal by the pilot FMBR have demonstrated strong performance. 16S MD DNA sequencing confirmed that the FMBR pilot system relied primarily on SND bacteria to remove nitrogen. SNDs require 20-30% less oxygen and 40% less C than most other N bacteria. This resulted in an energy saving of 77%. The high abundance of SND and DPAO bacteria, which have stronger endogenous respiration, reduced sludge production by 50%. Combined with other factors, the annual volume of biosolids requiring off-site disposal has been reduced by 65%. Ultimately, DNA and operational data confirmed the results – the simultaneous removal of C, N, and P, in a single reservoir with a surprisingly low amount of energy, footprint, and biosolids waste.

fermenting bacteria


The references

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

2. Xu, D., Liu, S., Chen, Q. et al. “Compositions of the microbial community in different functional areas of the 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 bacteria Pseudomonas sp. DNA-42. Appl Biochem Biotechnol 175, 2000-2011 (2015).

5. Bergey’s Manual of Systematic Bacteriology, 8th edition, by RE Buchanan and NE Gibbens, page 517.

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

7. Lv, Xiao-Mei et al. “A Comparative Study of the Bacterial Community in Denitrification and Traditional Biological Improved Phosphorus Removal Processes.” Microbes and environments vol. 29 (3): 261-268. (2014).

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