Wastewater Treatment and Biofuels, Part 2: Study Results and Implications

This blog post is based on my recent paper in the Journal of Environmental Engineering, the first of three that will ultimately be published from my PhD research, entitled Populus Species Mitigate Nitrogen Breakthrough in Wastewater Infiltration Systems for Year-Round Treatment and Recovery

The circular economy challenges industries to consider all phases of a product's lifecycle, from materials acquisition to disposal at end-of-life, as well as the other waste streams created in the manufacturing process. The biofuels industry is one which is taking steps toward incorporating more circular economy concepts into its process design.

A research group at the University of Washington proposed a biorefinery that would integrate biomass production, processing into biofuels, and wastewater treatment onsite. The investigation unlocked a number of avenues for further study, including my own PhD research on land application treatment. I studied the process of using biofuel crops to remove nutrients from wastewater while also producing biomass in the form of hybrid poplar trees.

Last week in Part 1 I discussed in detail the motivation for our experiment and how it fit into the grander design of the integrated biorefinery. I also described the set-up and the experimental plan. Today, I'll reveal the results of our study, the key conclusions that we were able to draw, and how we'll apply this information going forward as we continue to push for a circular approach to biofuel production!

Experimental Set-up: Our Reactors

Our experimental reactors simulating land application treatment, located at the University of Washington's Center for Urban Horticulture, consisted of three treatment groups:

• Planted reactors with poplar trees, irrigated with wastewater, to test treatment capacity

• Soil-only reactors with no trees, irrigated with wastewater, to show the effects of the trees on wastewater treatment

• Control reactors with poplar trees, irrigated with tap water, to show the effects of the wastewater on trees

We irrigated the reactors and let the trees and soil treat the water. Here are some pictures taken over the course of the study to show you how the set-up worked. We applied wastewater at the soil surface with drip irrigation lines.

The water then moved through the system, where it encountered soil microorganisms and, in the reactors with trees, tree roots and associated microorganisms.

Finally, we collected the water from ports on the bottom and tested it for nitrogen and carbon content.

Results - Nutrients

We observed a >90% decrease in total nitrogen in the water treated by reactors with trees. The nitrogen concentration in almost all samples was below legal discharge limits, meaning it could be released to the environment after treatment just like water leaving a traditional wastewater treatment plant.

This was in contrast to the soil-only reactors. They had much poorer performance in nitrogen removal, ranging from around 70% in the active spring and summer months, to less than 10% removal during the winter. In the later experiments when nitrogen content in the wastewater was increased, removal even during the summer months was only 61%.

We also considered a specific form of nitrogen, nitrate, in our analysis. Nitrate is an important pollutant because it can cause negative human health effects, especially in infants and young children. Like with total nitrogen, nitrate removal was much greater in reactors with trees. Planted poplar reactors removed >90% of the nitrate in most samples, while soil-only reactors removed very little nitrate. In fact, in many samples from soil-only reactors, there was more nitrate in the water coming out than the water going in. This suggested that the other forms of nitrogen in the wastewater, such as ammonia and organic nitrogen, were converted to nitrate by microorganisms. Then, the nitrogen cycle stopped due to one of a few potential limiting factors, resulting in nitrate buildup. The most likely limiting factor was the low content of organic carbon, which is needed for nitrate transformation, in the soil.

Organic carbon removal from wastewater was consistently above 90% throughout the experiment in both poplar and soil-only reactors, reducing concentrations from about 80 mg/L to below 10 mg/L to meet discharge standards. In winter, COD removal was less efficient in reactors both with and without trees, and the discharge standard was not met in either treatment. In the final season of operation, we included high levels of organic carbon in the wastewater to push the limits of the system. We found that the reactors with trees could handle up to 500 mg/L of carbon. This is much higher than the carbon content we would see in the partially-treated wastewater that is typically applied in land application systems.

Soil Properties and Leaf Nutrients

Soil moisture was consistently between 15% and 19% throughout the experiment. This told us that the soils were well-drained and that there were likely both aerobic and anoxic environments in the soil, providing capacity for the entire nitrogen cycle. This conclusion about the soil environment aligned with the nitrogen removal results in planted poplar reactors, which showed evidence of both aerobic ammonia transformation and anaerobic nitrate transformation. It also suggests that lack of anoxic environments was not the cause of limited nitrate transformation in soil-only reactors, and supporting the conclusion that limited organic carbon was instead the culprit.

We were surprised to find that the organic carbon removed from the wastewater did not build up in the soil as soil organic matter. From other studies, we expected to see soil organic content increase over time (Becerra-Castro et al. 2015). The fact that it didn't told us that our entire system, even with trees to provide extra carbon through their roots, was starved for energy. All of the carbon was used immediately for energy instead of being stored for later. 

One reason for this could be that we used soil with very little carbon in our initial setup, so the trees and the soil microbes did not have any reserves to work with and had to use the carbon as it came in. It might also reflect one of the key limitations of the study - the microbes and the trees were separated from the rest of the environment. At a real biorefinery, the trees would be planted directly in the ground, where they could draw nutrients from the larger soil pool around them instead of being limited to only the nutrients we added in the wastewater.

The leaves on treated poplar trees compared to leaves on tap water control trees was one of the clearest indicators of difference in our study. Below, leaves from treated trees are shown on the right, and leaves from control leaves are on the left.

The leaves from treated reactors were over 3x larger on average than those from control trees. In addition to being nearly the size of my face, the treated leaves were thicker, greener, and healthier overall than control leaves. We believe the leaves may have acted as a nitrogen sink, a repository where trees stored the nitrogen from the wastewater, and used the increased leaf surface area to generate more biomass via photosynthesis.

Key takeaways from our research

We learned three important things from our research on poplar land application for wastewater treatment and biomass production:

1. The trees were key components in nutrient removal from the wastewater

Nitrogen removal performance was significantly better in reactors with trees compared to those without. This was the expected result, but observing that result in hundreds of samples over 18 months of study was reassuring.

The observed nitrogen removal by poplar trees also provided important evidence for stakeholders who might be involved in the decision to create an integrated biorefinery, or for anyone interested in land application as wastewater treatment. It suggested that adding trees can provide additional filtration capacity to land treatment systems.

2. The trees took up the nutrients and used them for growth

This was clear from a visual assessment of the trees, including obvious differences in leaf size, height, and trunk thickness. The trees on the left in this picture received tap water, and the trees on the right received the wastewater.

But because we're scientists, we also ran some statistical tests. The trees that we watered with wastewater were 5 times larger by mass than the trees watered with tap water, with all other environmental conditions being identical. Because of this size difference, total leaf mass on treated trees was also much greater than control trees. This suggested to us that the nutrients from wastewater were taken up by trees and used to create both woody (trunk) and leaf biomass. From the perspective of the biorefinery, this finding suggested on-site land application treatment as viable a way to recycle nutrients from wastewater into additional poplar tree biomass and increase overall revenue.

3. Trees may allow land application treatment systems to operate year-round

One limitation to land application wastewater treatment is that in most locations, water is only applied during the growing season. When the trees are dormant, wastewater is transported to traditional offsite facilities for treatment. In cold climates, winter operation isn't possible anyway, because the applied water freezes before it can move through the soil. In these environments, some facilities store the wastewater in the field as layers of frozen ice, which then melts and is treated in the spring, fueling early-season growth of poplar trees (Khurelbaatar et al. 2021).

In temperate climates like the Pacific Northwest, which experience freezing temperatures only occasionally, we have the physical capability to operate a poplar wastewater treatment system year-round. The limitation here is regulations that permit application only during the growing season. There is certainly logic behind this limitation. In the winter, trees stop growing, and soil processes slow down, leading to a decrease in nitrogen and carbon cycling activity. This leads to worries that nutrients will not be fully removed from the wastewater as it moves through the soil, thus releasing pollution into the environment.

Our results challenged this assumption that dormancy requires pausing wastewater treatment. We found that even when trees weren't growing, the microbial populations on the roots maintained significant nitrogen removal activity. This meant that nitrogen was still removed to below legal discharge limits during the winter months, indicating the potential for successful treatment in cold weather. Importantly, the same was not observed in systems without trees. Without the roots providing the structure and habitat for microbial populations, the microbes alone were not sufficient to remove nutrients to below legal discharge limits.

Conclusion

Together, our results and key takeaways tell an important story for practitioners considering land application for wastewater treatment. Including vegetation in a land application system can improve the treatment capacity, both increasing nutrient removal potential and expanding the available seasons of operation, while also producing biomass that, in the case of an integrated biorefinery, can be used on-site.

One of the central tenants of the circular economy is transforming waste into usable products. Our study highlighted the potential for recovery and transformation of waste in two ways. Nutrient recovery, in which nutrients are converted into tree biomass, was shown by the significant increases in the size of poplars that received wastewater. Water recovery potential was shown by the effective removal of nitrogen and carbon from the wastewater to below discharge standards in the poplar reactors. This suggested that a biorefinery could collect the water after it passes through a land application treatment system and use it directly for additional processes, which saves water resources and also provides financial savings.

While plans to build the integrated biorefinery in central Washington have been put on hold, the biofuel market is still expanding, slowly but surely. I believe biofuels have an important role to play as a green energy source in a climate-conscious future.

This study of nutrient fate in land application wastewater treatment was only one part of my PhD work. I also investigated the microbial communities in the soil that were responsible for nutrient transformation, and quantified abundance of certain genes that are important in the nitrogen cycle. These manuscripts are currently under preparation for submission, and I can't wait to tell you all about the work after it is published!

Want to learn more about how microbiology intersects with the biofuels industry? Interested in incorporating land application wastewater treatment into your manufacturing process? Reach out for a free consultation today!

References

Becerra-Castro, C., Lopes, A. R., Vaz-Moreira, I., Silva, E. F., Manaia, C. M., & Nunes, O. C. (2015). Wastewater reuse in irrigation: A microbiological perspective on implications in soil fertility and human and environmental health. Environment International, 75, 117-135. doi:10.1016/ j.envint.2014.11.001

Kargol, A. K., Montes, D., Dahal, S., and Gough, H. L. 2025. "Populus Species Mitigate Nitrogen Breakthrough in Wastewater Infiltration Systems for Year-Round Treatment and Recovery." Journal of Environmental Engineering 151(12): 04025082. doi:10.1061/ JOEEDU.EEENG-8180.

Kargol, A. K., Burrell, S. R., Chakraborty, I., and Gough, H. L. 2023. "Synthetic wastewater prepared from readily available materials: Characteristics and economics." PLOS Water 2(9). 10.1371/ journal.pwat.0000178.

Khurelbaatar, G., van Afferden, M., Sullivan, C. M., Fühner, C., Amgalan, J., Londong, J., & Müller, R. A. (2021). Wastewater Treatment and Wood Production of Willow System in Cold Climate. Water, 13(12). doi:10.3390/w13121630