The excessive use of fossil fuels, industrialization, and growing populations have increased environmental pollution, global warming, and global energy demands (Dimitriadis & Bezergianni, 2017). Due to these problems, a new alternative energy source must be used. Biomass conversion to liquid transportation fuels is a major category of renewable energy, with various waste biomasses as leading candidates (Dimitriadis & Bezergianni, 2017). Multiple thermo-chemical conversion (TCC) technologies exist to convert biomass into fuels with one of the most promising methods being hydrothermal liquefaction (HTL). The main benefit of HTL is that it does not require that the biomass be dried before processing which would be a fuel- and cost-intensive process (Grande et al., 2021; SundarRajan et al., 2021). However, HTL produces toxic solid, gaseous, and aqueous byproducts. The hydrothermal liquefaction aqueous-phase byproduct (HTL-AP) is thought to be the factor limiting commercial viability of the HTL process, particularly because some of the constituents have inhibitory effects on nitrifying bacteria (Macêdo et al., 2023). There are existing treatment technologies for HTL-AP such as anaerobic digestion, wet air oxidation (WAO), and more. The simplest treatment method at a wastewater treatment plant (WWTP) using HTL for sludge disposal would be to return the HTL-AP to the v headworks to be treated alongside standard wastewater flow (MBE, 2019). However, the inhibitory effects may cause the WWTP to not meet ammonia regulations, if relevant. In this project, four 3.5-L bench-scale aerated reactors were built to simulate conventional activated sludge wastewater treatment to develop methods to determine the long-term effects of HTL-AP on nitrification. Two reactors were controls fed only synthetic wastewater, and two reactors were fed synthetic wastewater with a small dilution of HTL-AP, called AP reactors. Two different doses of hydrothermal liquefaction aqueous phase (HTL-AP) were tested on long-operated activated sludge systems. A 275x dilution factor (DF) of HTL-AP was tested for 132 days and a 2750x DF was tested for 23 days. The weekly nitrogen data (total ammonia nitrogen, nitrate, and nitrite) suggested that both doses inhibited nitrification in the bench-scale activated sludge systems. Additionally, the nitrifying bacteria did not adapt over time to be able to nitrify the wastewater with HTL-AP. During the first experiment with a 275x DF, no nitrate was detected in the AP reactors (/L NO3 - -N) and nitrite was sparsely detected. The control reactors had low concentrations of nitrate of around 3-8 mg/L NO3 - -N and high concentrations of nitrite, typically around 10-40 mg/L NO2 - -N. Therefore, the AP reactors were performing minimal nitrification, if any, while the control reactors were primarily performing partial nitrification. Before implementing the 2750x DF of HTL-AP, all reactors were emptied, cleaned, and restarted with nitrifying mixed liquor from the San Luis Obispo Water Resource Recovery Facility membrane bioreactor (SLO WRRF MBR). By the end of the 23 days of HTL-AP exposure at a 2750x DF, the treatment reactors had a sharper nitrate decline with final NO3 - -N concentrations around 1 mg/L compared to about 5-7 mg/L vi NO3 - -N in the control reactors. At this time, nitrite concentrations were not considerable in any reactor. Because nitrification was not ideal even in the control reactors, future work is advised, including stabilizing the solids retention time and changing the synthetic wastewater formula to support higher concentrations of treatment bacteria. Some methods for the specific oxygen uptake rate (SOUR) test that are not explicitly or extensively outlined in ISO 8192 (ISO, 2007) were examined. First, a 10- minute trial duration was deemed sufficient compared to 20 minutes (R 2>0.99 in either case and Preliminary SOUR tests using the correct methods found that raw HTL-AP doses of 50x, 125x, and 250x dilution factors (DF’s) inhibited nitrification by about 80 to 105% in nitrifying sludge from the SLO WRRF MBR. Raw HTL-AP doses of 500x and 1000x DF’s did not inhibit nitrification respiration rates. From a SOUR test on this same sludge but on a different day, HTL-AP pretreated by wet air oxidation (WAO) had near-zero vii (neg. 0.9%) inhibition on nitrification respiration rates at a 250x DF compared to the 66% nitrification inhibition found in raw HTL-AP at this 250x DF on this testing date.