Overcoming Anammox Challenges


Using CRISPR to Establish Transgenic Derivatives of Anammox Bacteria


Anammox (anaerobic ammonium oxidation) bacteria is one of the novel microorganisms used for the removal of nitrogen contained in wastewater. We intend to induce genetic editing by introducing CRISPR gene scissors to anammox bacteria. Ultimately, we want to modify wild-type species with improved treatment efficiency (i.e. have a high growth rate or the ability to grow in low temperature) compared to the standard anammox bacteria. 


  • Explore the pure culture conditions of the industrial-level anammox bacterial species

  • Secure functional gene sequences via high-throughput sequencing

  • Develop CRISPR gene editing platform to specifically target anammox functional genes

  • Establish a way to transfer CRISPR gene scissors into the anammox cell

  • Ultimately, establish a way to edit desired functionality through introducing CRISPR gene scissors to the anammox genome strain


Internationally, there is a growing interest in water treatment technologies due to strict regulations on water quality.  More specifically, there is a growing demand on the environmentally friendly technologies for significantly reducing energy consumed by biological treatment of nitrogen in sewage and wastewater. Anammox bacteria has attracted much attention in the field of wastewater treatment due to its ability to reduce nitrogen contents to dinitrogen gas in an anaerobic setting while maintaining a low energy usage and low operating cost.Despite having many advantages, anammox technology is often limited by cold temperature and slow rate of cell division. Because of these limitations, there needs to be technological differentiations to make anammox technology a viable treatment solution.

Since its successful development in early 2013, genome editing using CRISPR / Cas system gene scissors has advanced dramatically in recent years with applications in various fields. CRISPR gene scissors technology is a revolutionary tool for editing specific genes, and is expected to induce the desired functional transformation when applied to anammox bacteria. NGS (next generation sequencing) for genome analysis will be performed after making and applying the CRISPR gene editing platform to the anammox bacteria.  There is a worldwide competition for improving functions in bacterial strains for wastewater treatment technology. By introducing CRISPR gene editing tool to anammox genome, we expect to make a highly versatile transformant that is not limited to industrial use. 


Strategy 1.
Establishment of optimized culture condition for Anammox bacteria

  • Anammox strain has become largely used in industrial sector, but it must be cultured as a highly enriched strain for genome editing.

  • Establish optimal culture conditions for pure culture strain

Strategy 2.
Acquire functional gene sequences to target in Anammox bacteria

  • When a gene is mutated in a demonstration case, target easily observable genes when they are expressed (i.e., ones that either change colors or react to specific chemicals)

  • Investigate target gene’s nucleotide sequences through databases such as literature and NCBI

  • Obtain the genome sequences in the present strains through next-gen DNA sequencing

Strategy 3.
Making gene scissors for target genes & transforming the scissors to cells

  • Produce a CRISPR gene scissors that can target gene sequences obtained in strategy 2

  • Create at least four CRISPR gene scissors for each gene to determine the most efficient gene scissors

  • Establish a way to transform the produced CRISPR gene scissors most efficiently into cells (make various attempts such as electroporation and lipofectamine on the carrier)

  • Anamox strains which gene scissors have been introduced to are screened for transformants through NGS sequencing

  • Obtain transformants that have been knocked out by CRISPR


  • Using the CRISPR gene scissors to produce transformants in Anammox is expected to be revolutionary in the process of environment-friendly water treatment

  • In the future, apply CRISPR gene scissors to not only a single gene but also several gene families related to bacterial growth

  • Through screening experiments, produce highly efficient anammox transformant.


Bio-Algae Nutrient Removal (BANR)

The Bio-Algae Nutrient Removal (BANR) process is a GRI patented technology that allows nitrification and algae growth to occur in a single reactor. The dissolved oxygen necessary for wastewater treatment using the BBF is supplied through the byproducts of accelerated algal growth, for which the BANR process has overcome the limitation of sunlight dependency by employing LED light in the absence of sunlight.

BANR is a part of several new emerging technologies. Different species of algae are utilized in nitrigen and phosphorus removal as well as for energy neutralization in wastewater treatment plants. This is considered an effective organic pretreatment (BBF). Results of using BANR include CO2 reduction and resource recovery through algae production and reuse.

BANR integration

  • Zero Energy & Zero Chemical consumption

  • Energy production through carbon diversion and biomass processing

  • By removing not only solid organics but also soluble organics, BBF ensures that new energy saving processes such as OBA and BANR, can work properly alongside WWF



AOBs, AMXs, and denitrifiers coexist and organic removal, conventional denitrification, and Anammox reaction are simultaneously carried out in one reactor. 

Using a membrane diffuser, supply oxygen needed only for nitritation

  • Denitrifiers (XHD) denitrify NO2 and NO3 using organic matter in influent

  • AMXs can be used to remove nitrogen through single-nitrogen reaction with NO2 generated by AOB from NH4 in the influent.

  • Maximize oxygen transfer rate (OTE 90-100%) to save energy


  • Supply only the amount of oxygen required for nitritation

  • Supply inorganic carbon required for nitrification and anammox reaction

  • Supply gas volume required for reactor agitation and biofilm thickness control