Urine-enriched biochar: Coupling sustainability in sanitation and agriculture

Nitrogen fertilizers are frequently over-applied to agricultural fields, leading to the pollution of water systems, the release of the powerful greenhouse gas (GHG) nitrous oxide, and stratospheric ozone depletion (Zhang et al., 2015). Additionally, fertilizers are not always accessible to smallholder farmers, critical actors of the global food system (Rapsomanikis, 2015), and fertilizer prices are subject to volatility (Crespi et al., 2022). These global issues highlight the need for a circular approach to nutrient management that prioritizes recovery and reuse, particularly in a world with a changing climate.

One largely unused resource for nutrient recovery and reuse is human excreta. Once humans reach adulthood, we excrete nearly all the N, phosphorus (P), and potassium (K) that we consume (Jönsson et al., 2004). Human urine is an important potential waste stream for resource recovery, as it contains most of the N, and approximately half of the P and K that we excrete (Harder et al., 2019). A paradigm shift in which human excreta is treated as a resource rather than pollutant could lead to a transformation in nutrient management (Bouwman et al., 2009). Container-based sanitation (CBS) is an emerging technology in which human excreta is source-separated in containers and transported to a local facility for waste treatment, processing, and reuse (Russel et al., 2019). This study explores urine-N recovery scenarios relevant to CBS systems. We use biochar, a low-cost carbon-rich adsorbent, to recover urine-N from human urine stored in conditions realistic to CBS and other urine-diverting systems. To the best of our knowledge, this research is the first to examine multiple types of urine-enriched biochar (UEBC) made with various kinds of urine and biochar in a single greenhouse growth trial. Figure 1 summarizes our research approach and how it relates to CBS systems.

As of 2020, 4.5 billion people lack access to safely managed sanitation, of which 2 billion have no access to basic sanitation (United Nations, 2020). United Nations Sustainable Development Goal (SDG) 6 aims to ensure access to clean water and sanitation for the global population by 2030 (United Nations, 2020). Unless current rates of implementation increase considerably, SDG 6 will not be met by the target date (United Nations, 2020). CBS has the potential to help bridge this gap in global sanitation provision by providing resilient, low-to-no water, decentralized sanitation with the added benefit of nutrient recovery and reuse (Tilmans et al., 2015). CBS is commonly employed in areas where conventional sewerage systems face challenges, such as densely populated low-income urban settlements and regions prone to water contamination from flooding or high water tables (Russel et al., 2019). CBS systems typically collect feces and urine in separate, above-ground containers that can be sealed and transported for treatment and/or recovery.

Management in CBS systems usually focuses on the safe treatment and reuse of feces for various outcomes (Figure 1). Feces-derived end-use products include compost, animal feed, and biomass/biogas fuels (Russel et al., 2019). However, recovering nutrients from both urine and feces would better close local nutrient cycles (Ryals et al., 2021). Nitrogen in urine is plant-available, present as mainly urea (75%–90%), and the remaining N as NH₄⁺, creatine, or nitrate (Jönsson et al., 2004; Rose et al., 2015). The P and K in urine are excreted in plant-available ionic forms. Notably, these are some of the most common forms of N, P, and K found in synthetic fertilizers (Jönsson et al., 2004). Though urine has been used as a fertilizer, both in ancient agricultural practices (Angelakis et al., 2018) and in recent scientific research, its adoption in modern agricultural systems is slow (Karak and Bhattacharyya, 2011). Proper urine treatment in CBS systems is also important to reduce nitrate and pharmaceutical leaching to groundwater, particularly in places with high water tables (Russel et al., 2019).

A major barrier to urine-nutrient recovery in CBS systems is the difficulty and cost of transporting large quantities of liquid (Russel et al., 2019). Additionally, while the mass of excreted nutrients is higher in urine than feces, the concentration of these nutrients is lower since urine is 97% water (Senecal and Vinnerås, 2017). This barrier points to the need to concentrate urine-nutrients at a decentralized scale into a solid product that would be easier to transport. While methods like NH₃ stripping, struvite precipitation, and electrochemical technologies have demonstrated successful nutrient recovery from urine (Kabdaşlı and Tünay, 2018), they may have high operational costs and may not be suitable for implementation at a decentralized scale (Kundu et al., 2022). In our research, we explore an alternative approach for urine-N recovery: adsorption to biochar. Biochar, produced through biomass pyrolysis (Weber and Quicker, 2018), offers a potentially low-cost and widely available adsorbent option that can be produced and utilized in resource-constrained settings. Although other adsorbents may exhibit higher N removal efficiency (Tarpeh et al., 2017), we specifically investigate biochar as an adsorbent for urine-N due to its potential to repurpose wasted biomass, sequester carbon in soil when applied, and mitigate climate change (Masrura et al., 2020). Field trials have already demonstrated the efficacy of UEBC as a fertilizer (Schmidt et al., 2015; Schmidt et al., 2017; Sutradhar et al., 2021). Biochar could potentially be used to recover urine-N at the individual toilet scale (i.e., an attached filter) or the neighborhood scale (i.e., communal soak pit) (Ryals et al., 2021).

Many areas in UEBC research are underexplored. There is substantial variation in the chemical composition of urine due to storage conditions in CBS systems. Urease, a ubiquitous bacterial enzyme, hydrolyzes urea to NH₄⁺ and carbon dioxide (Krajewska, 2009). It is typically present in large quantities in urine storage systems and rapidly hydrolyzes urea in fresh urine (Tarpeh et al., 2018). Tarpeh et al. (2018) found lower NH₄⁺ concentration in open containers compared to closed containers in a CBS system, attributed to NH₃ volatilization. This has implications for the value of urine-derived fertilizers produced with urine from open containers, as gaseous loss of N as NH₃ may be significant. NH₃ emissions from open urine containers may also have consequences for human health, as NH₃ can react with N and sulfur oxides to form PM_2.5, which can cause severe respiratory and circulatory problems (Stokstad, 2014). Additionally, NH₃ emissions cause an unpleasant odor in urine storage systems (Hashemi and Han, 2017). Understanding how the various solution chemistry created by different urine storage conditions influences N adsorption to biochar is an important part of practical urine-nutrient recovery research. The mechanisms, kinetics, and adsorption affinity of organic and inorganic molecules to biochar have also been shown to differ across biochar feedstocks and pyrolysis temperatures (Ambaye et al., 2020).

The objective of this research was to assess the effect of urine storage conditions on N adsorption to different biochars, and the effect of these UEBCs on tomato growth and plant N uptake. We sought to identify how different urine-biochar combinations may impact agricultural outcomes. We produced UEBC from 3 urine types: fresh (hereafter Fresh); stored, covered urine (hereafter Stored); and CBS-style urine (hereafter CBS), and 3 types of biochar (sewage sludge, wood waste, and walnut shell), for 9 UEBC combinations. These UEBCs were compared with a synthetic and organic fertilizer, an unfertilized control, and urine-only treatments. We expected UEBC treatments to outperform urine-only treatments due to the adsorption and subsequent slow release of nutrients, as opposed to the gaseous and leaching N losses that likely reduce fertilization quality in urine-only treatments. We expected CBS treatments to lead to lower yield due to initial N losses as NH₃ during storage, and all treatments to outperform unfertilized controls.

Human urine was collected from consenting volunteers over age 18 from the University of California (UC), Merced. Collection protocols were approved for expedited review by the Institutional Review Board of UC Merced (IRB # UCM2020-171). Urine was provided by volunteers in 100 mL containers and immediately refrigerated until use, at most 1 week later. Three urine treatments were prepared: Fresh urine, Stored urine, and CBS urine. CBS urine was completely urea-hydrolyzed and left uncovered for 1 day in a CBS-style container to mimic storage conditions at a leading CBS provider, Sustainable Organic Integrated Livelihoods (SOIL) in Haiti.

Fresh urine was combined into multiple 1 L polypropylene containers. It was confirmed to be in the electrical conductivity (EC) range of fresh urine (approximately 11 ms/cm) based on prior experimentation and work by Ray et al. (2018). Urine to be urea-hydrolyzed for Stored and CBS treatments was combined in multiple 1 L containers. To account for differences in environmental enzyme loading in CBS systems, this urine was allowed to urea-hydrolyze completely before it was either refrigerated until use (Stored) or left uncovered (CBS). All containers were filled nearly completely to mitigate volatilization within the headspace while covered; 0.533 g/L of urease (CAS 9002-13-5, Fisher Scientific) was added to each container based on methods used by Ray et al. (2018) and shaken for 30 min at 180 oscillations/min (Eberbach E6010.00). All containers were kept sealed during urea hydrolysis. Urine was considered completely urea-hydrolyzed when the EC values stabilized at approximately 24 ms/cm, consistent with Ray et al. (2018) and prior experimentation, after approximately 36 h at room temperature. After urea hydrolysis, 3.18 L of urine was transferred to a 3.78 L CBS-style urine collection container (Figure S1) and left uncovered for a day to mimic CBS urine storage conditions at SOIL. This volume was chosen based on expert opinion at SOIL, stating that urine containers are typically allowed to fill approximately 3/4 of the way before being emptied (J Jeliazovski, personal communication, 08/07/2021). We recognize other urine storage conditions were dissimilar to SOIL’s CBS conditions, such as temperature and humidity, in our lab-scale study. A subsample of each urine type was frozen until later NH₄⁺ analysis using the microplate colorimetric salicylate-nitroprusside method (Mulvaney, 1996) (Agilent BioTek Gen5 Microplate Reader, Agilent Technologies, Santa Clara, CA, USA). Samples were thawed at room temperature immediately before analysis, and 0.533 g/L urease was applied to half of the Fresh urine sample and shaken for 30 min at 180 osc/min to determine urea content. All urine samples were run in triplicate.

Three biochar types were used in this study: a walnut shell biochar produced at 350°C by pyrolysis (hereafter Walnut Shell) (NextChar, Amherst, MA), a sewage sludge biochar produced at 550°C by pyrolysis (hereafter Sewage Sludge) (UK Biochar Research Centre, SS550), and a wood waste biochar produced at 593.3°C by gasification (hereafter Wood Waste) (Aries GREEN™ All Natural Soil Conditioner). Biochar pH and EC were measured using methods recommended by Balwant et al. (2017). Briefly, biochar was prepared at a 1:10 biochar:deionized water ratio, shaken for 1 h at 150 rpm, and allowed to settle for 30 min. Readings were taken in triplicate from each sample. Other physiochemical properties were obtained from the supplier or research partners. Biochar properties are shown in Table 1. All urine types were combined with each biochar for a total of 9 UEBCs. UEBC was prepared at a 200 g:1 L ratio. This ratio was chosen based on preliminary experimentation that found this ratio favorable for urine-N adsorption. Respective urine and biochar types were added to 1 L containers, briefly agitated, and allowed to soak for 48 h. All containers remained sealed for the duration. UEBCs were then allowed to drain freely over a 500-µm sieve and refrigerated until use. An effluent sample from each was filtered through a 0.45 µm polypropylene filter and frozen for later NH₄⁺ analysis. This aqueous phase N (mg urine-N g-biochar⁻¹) (Q_aq) was calculated as follows:

where C_in and C_f are the initial and final N concentrations in solution (mg L⁻¹), M is the mass of biochar (g), and V is the volume of urine (L). Q_aq includes all N removed from the urine, in both the >500-µm and <500-µm size fractions. A subsample of each UEBC was allowed to air dry for elemental analysis using a Costech 4010 Elemental Analyzer (Costech Analytical Technologies Inc., Valencia, CA). These data were used to determine the solid phase N (mg urine-N g-biochar⁻¹) (Q_s) in the >500-µm size fraction, as follows:

where %N_UEBC is the percentage total N in the UEBC, and %N_raw is the percentage total N in the unenriched biochar. The mass of N retained by the biochar particles <500-µm (mg-N) (M_<500-μm) was calculated as follows:

The portion of N potentially lost to volatilization is neglected. Separate subsamples of UEBC were oven-dried at 105°C until a stable weight was reached to determine moisture content.

A 5-week growth experiment was conducted to test the efficacy of UEBCs as fertilizers and investigate urine-N plant availability. Tomato (Burpee Early Girl) was chosen as a globally important crop with relatively rapid growth rates (Food and Agriculture Organization [FAO], n.d.). Fifteen treatments were tested: 9 UEBCs (Sewage Sludge-Fresh, Sewage Sludge-Stored, Sewage Sludge-CBS, Wood Waste-Fresh, Wood Waste-Stored, Wood Waste-CBS, Walnut Shell-Fresh, Walnut Shell-Stored, Walnut Shell-CBS), 3 urine-only treatments (Fresh, Stored, and CBS), a synthetic fertilizer (Miracle-Gro® Water Soluble Tomato Plant Food), an organic fertilizer (Jobe’s Organics Vegetable & Tomato Granular Plant Food), and an unfertilized control. All UEBCs were tested at application rates of 1%, 2%, 6%, and 10% (w/w) on a dry mass basis. For urine-only treatments, the volume of urine applied aligned with the corresponding UEBC application rate. This allowed us to test the same amount of urine with or without biochar. For example, at a 1% UEBC application rate, 0.107 g biochar and 0.535 mL urine were applied in the UEBC mixture. Thus, for the 1% urine-only application rate, 0.535 mL urine was applied alone; 1.07 mL of urine was applied at the 2% application rate; 3.21 mL at the 6% application rate; and 5.35 mL at the 10% application rate. These urine-only treatments are hereafter referred to as “Fresh 1%,” for example. The organic and synthetic fertilizers were applied at the start of the study at a rate of 150 kg-N/ha, the upper N fertilization rate suggested for tomatoes (FAO, n.d.). All other N application rates were determined post hoc (Table 2). Amendments were incorporated into a soilless substrate (Premier Tech Pro-Mix BX Mycorrhizae) at the appropriate ratio. The soilless substrate is 79%–87% sphagnum peat moss and 10%–14% perlite mixture inoculated with Glomus intraradices (Premier Tech Horticulture, Québec, Canada). 10.7 g of amendment + soilless substrate was applied to 90-cm³ plug flat pots, with 9 replicates per treatment, for a total of 459 plants. Trays were brought to 90% of field capacity, determined by weight and preliminary experimentation. One tomato seed was sown into each pot. Pots were maintained at 90% of field capacity by misting with an automated sprayer or watering with a watering can, typically daily. Deionized water was used to prevent the introduction of nutrients from tap water.

Plant height was measured every 4 days starting 8 days after first emergence for 6 randomly selected plants per treatment. Height was measured with a ruler from the soil surface to the petiole of the highest leaf. The number of leaves >1 cm was also recorded. Final plant height, number of leaves >1 cm, and leaf area of the largest leaf was measured for all plants after 5 weeks. Leaf area was measured with a Tamaya Technics Planix 5 Digital Planimeter. In the case that the largest leaf had very irregular margins, the second largest leaf was measured. After final plant growth monitoring, all plants were destructively harvested. Above-and-belowground biomass was separated at the soil surface. Soil was removed from belowground biomass using deionized water. Biomass samples were dried in an oven at 65°C until a stable weight was reached. Above-and-belowground biomass for each treatment (n = 5) were ground separately in a ball mill (SPEX SamplePrep 8000 M Mixer/Mill®, SPEX SamplePrep, Metuchen, NJ, USA). All replicates were ground together for each treatment for one analysis per treatment (above or below). Samples were subsequently analyzed for elemental C and N content.

All statistical analyses were performed in R version 4.1.3. All tests used a significance level of 5%. Separate three-way ANOVAs were performed for total biomass, aboveground biomass, belowground biomass, final plant height, final number of leaves >1 cm, leaf area, and final leaf number as the response variable and biochar type, urine type, and application rate as factors. Data were log transformed prior to analysis. Post hoc Tukey’s honestly significant difference (Tukey HSD) tests were performed with the “HSD.test” function in the agricolae package. A one-way Kruskal–Wallis test with a Bonferroni correction was performed to compare N content of all UEBCs with the “kruskal” function in the same package. A general linear model was used to compare final germination percentage across treatments. One-and two-way ANOVAs were also performed to compare grouped data, with post hoc Tukey’s HSD tests using the “TukeyHSD” test in the stats package. Separate one-way ANOVAs were performed for urine, biochar, and application rate as predictor variables and total biomass as the response variable. To compare the difference between UEBCs and urine-only treatments, we performed a two-sample Wilcoxon Rank Sum test using “biochar/no biochar” as grouping terms, using the “compare_means” function in the ggpubr package. Two-way ANOVAs were performed with total biomass as a response variable and all combinations of biochar type, urine type, and application rate as factors to test for interaction effects. Linear regression models were fitted to N data within each biochar type (or no biochar, referred to as “urine-only”) with mg urine-N applied per pot as the predictor variable and mg-N uptake per plant as the response variable, using the ggpmisc package. A two-sample Wilcoxon Rank Sum test was used to compare Q_aq and Q_s, within biochar and across urine types.

Q_aq was significantly higher than Q_s across UEBCs (P < 0.001, Wilcoxon Rank Sum test) (Figure 2). Across biochar types, Q_aq indicates that 85%–98% of the initial N in urine regardless of type was removed from solution (Figure 2). M_{<500 μm} is shown in Figure S2. Q_aq shows that 25%–45% more N was removed from Stored than Fresh urine, and 25%–50% more than CBS urine (Figure 2). This discrepancy is likely due to Q_s being determined from analysis of biochar particles >500-μm. Previous studies have shown that larger biochar particle size fractions retain significantly less NH₄⁺ and take longer to reach equilibrium with NH₄⁺ (Kizito et al., 2015; Bai et al., 2018). The principal chemical differences between Fresh urine and Stored/CBS urine are their nitrogenous species, pH, and EC. Urea is the primary solute in Fresh urine, constituting 75%–90% of N in solution (Rose et al., 2015). Most N in Stored or CBS urine is present as NH₃/NH₄⁺ due to the complete activity of the urease enzyme in these urine types prior to UEBC preparation. The decrease in sorption capacity with increasing particle size may be due to the higher surface area and shortened diffusion paths of smaller particles. Specifically, small size fractions have a higher concentration of oxygen containing surface functional groups, and more sorption sites relative to larger particles (Nocentini et al., 2010). Thus, the difference between Q_aq (14–20 mg-N g-biochar⁻¹) and Q_s (1–4 mg-N g-biochar⁻¹) may be explained by particles <500-µm having more surface functional groups and containing the majority of N removed. Future work should determine N capacity in <500-µm particles via elemental analysis. The feasibility of recovering this small UEBC size fraction for use should also be explored. Future work should also investigate the role of pH in urine-N sorption to biochar. While monitoring the urea hydrolysis process, we found that the initial pH of Fresh urine averaged at 6.06 ± 0.28 and pH after urea hydrolysis averaged at 9.00 ± 0.15. The EC prior to urea hydrolysis was 11.40 ± 0.79 ms/cm, and EC after completion was 24.58 ± 0.63 ms/cm. These parameters are typical of fresh and urea hydrolyzed urine found in the literature (Karak and Bhattacharyya, 2011).

A different trend was seen with Q_s, where Fresh led to the highest removal of N (Figure 2). Q_s was significantly higher in UEBCs prepared with Fresh urine, compared with CBS or Stored urine for Sewage Sludge and Wood Waste (P < 0.001, one-way Kruskal–Wallis test) (Figure S3). Q_s for Sewage Sludge-Fresh was 571% and 357% higher than Q_s for Sewage Sludge-CBS and Sewage Sludge-Stored, respectively. Q_s for Wood Waste-Fresh was 1,377% and 473% higher than Q_s for Wood Waste-CBS and Wood Waste-Stored, respectively. The same trend held for Walnut Shell but was not significantly higher when compared across all UEBCs. Q_s for Walnut Shell-Fresh was 27% and 60% higher than Walnut Shell-CBS and Walnut Shell-Stored, respectively. Q_s was similar in Fresh UEBCs across biochar types. Sewage Sludge-Fresh averaged 3.76 ± 0.95 mg-N g-biochar⁻¹, Wood Waste-Fresh averaged 3.36 ± 0.21 mg urine-N g-biochar⁻¹, and Walnut Shell-Fresh averaged 3.94 ± 0.16 mg urine-N g-biochar⁻¹. Walnut Shell biochar had the highest Q_s across urine types, with an average of 3.09 ± 0.28 mg urine-N g-biochar⁻¹ for Walnut Shell-CBS, and 2.47 ± 1.73 mg urine-N g-biochar⁻¹ for Walnut Shell-Stored.

The differences between Q_s and Q_aq may also be attributed to preferential binding of urea molecules in Fresh urine to larger biochar particles. Alternatively, it is possible that urea has similar affinities for biochar particles in either size fraction. If this is the case, it may indicate a stronger affinity for urea molecules than NH₃/NH₄⁺ molecules for biochar surfaces, or that solution conditions in Fresh urine, such as lower pH and EC, are generally more favorable for adsorption to biochar compared to the higher pH and EC of Stored/CBS urine. It is possible that the lower EC in Fresh urine results in less competitive sorption of urea with other molecules, and thus a greater mass of bound urea compared to NH₃/NH₄⁺ in the Stored/CBS UEBCs. Solanki and Boyer (2019) studied the removal of pharmaceuticals from urine using biochar and found that adsorption of paracetamol from real urine treatments was significantly lower than from synthetic urine, which they attributed to the presence of metabolites in real urine. Though urea and paracetamol are vastly different molecules, this research suggests that competition between urine metabolites may reduce organic molecule adsorption to biochar.

Multiple studies have suggested urea uptake onto biochar or activated carbon is dominated by physisorption (Ganesapillai and Simha, 2015; Ganesapillai et al., 2016; Kameda et al., 2017). These studies agree that urea sorption to these adsorbents is governed by pseudo-second order kinetics. Simha et al. (2016) propose that urea adsorption to biochar is limited and controlled by intraparticle and surface diffusion. NH₄⁺ adsorption to biochar is believed to be controlled by cation-exchange, surface complexation with oxygen-containing functional groups, hydrogen-bonding, precipitation, or electrostatic interaction (Cai et al., 2016; Cui et al., 2016). Tarpeh et al. (2017) found that ion exchange was the dominant adsorption mechanism for NH₄⁺ in real, undiluted, urea hydrolyzed urine to biochar, among other adsorbents. Future research should develop adsorption isotherms and investigate adsorption mechanisms of urea, NH₄⁺, and NH₃ in Fresh, Stored, and CBS urine.

There was differential uptake of N in plant tissue across treatments (Figure 3). There is a strong positive linear relationship between urine-N applied and N uptake in plant tissue in the urine-only treatments, regardless of urine type (R² = 0.98) (Figure 3). This indicates that N was potentially still a limiting nutrient, as no plateau in N uptake in plant tissue is seen in the data. Even at 22.69 mg-N applied at the Fresh urine 10% equivalent volume application rate, 12.18 mg of N is found in the plant tissue. From the slope of the regression line, we can infer that about 30% of N applied in urine-only treatments is taken up in the plant tissue. Since we do not have isotope tracer or dilution data, we cannot be certain that the N in the plant tissue is the same as that applied to the pot. However, some urine-N is likely present in the plant tissue, as urine-N is excreted in plant available forms, regardless of urine type (Jönsson et al., 2004). It is likely that some of the urine-N applied was lost as gaseous-N such as NH₃ or nitrous oxide or leached as nitrate after soil microbial transformations. Similarly problematic N losses are found in synthetic N fertilizer application (Zhang et al., 2015), and gaseous and leaching losses have been previously demonstrated with urine applied as fertilizer (Kirchmann and Pettersson, 1994; Wachendorf et al., 2005). It is also likely that some urine-N is immobilized in soil microbial tissue. Similar trends were observed in N application and uptake for both above-and-belowground biomass (Figures S4 and S5). Linear regressions do not fit the UEBC N uptake data as well as for urine-only. Walnut Shell UEBCs exhibit a positive linear trend (R² = 0.44). Our results show that urine-N in Stored and CBS urine adsorbed more readily to Walnut Shell than Wood Waste or Sewage Sludge (Figure S3). Our results also imply that urine-N desorbed from Walnut Shell biochar more easily, regardless of urine type (Figure 3). This trend may be explained by the sorption/desorption behavior of surface functional groups on Walnut Shell biochar. This biochar was produced at 350°C, a relatively low pyrolysis temperature that is known to maintain oxygen-containing surface group functionality (Rasse et al., 2022). Negatively charged functional groups on biochar have shown optimal adsorption capacity for NH₄⁺ and urea (Masrura et al., 2020). This may explain the unique behavior of Walnut Shell biochar compared to Wood Waste or Sewage Sludge, as these were produced at approximately 593°C and 550°C, respectively.

Similar plant N uptake is seen across all application rates of Sewage Sludge-CBS and Stored UEBC, and Wood Waste-CBS and Stored UEBC. It is notable that significantly less N was applied with these UEBCs compared to Fresh UEBC in the same biochar groups. Urine-N applied ranges from 0.02 to 0.88 mg urine-N pot⁻¹ for Sewage Sludge and Wood Waste CBS and Stored UEBCs across application rates. However, plant N uptake ranges from 4.40 to 7.27 mg-N uptake plant⁻¹. This suggests a soil N priming effect, or native soil N turnover in response to N addition (Kuzyakov et al., 2000). It is clear the N in the plant tissue cannot only derive from Wood Waste CBS/Stored UEBCs, as the total N (biochar N + urine N) is lower than the plant uptake for application rates 1% and 2% (Table 2). It is also unlikely that much biochar-N is mineralizable. Fiorentino et al. (2019) observed low mineralization of biochar-N and a native soil N priming effect with the coaddition of urea and wheat straw biochar. While Sewage Sludge-CBS and Stored UEBCs had a higher total N content due to the high N content of the raw Sewage Sludge biochar (Table 1), it is unlikely that much of this biochar-N is mineralizable in the short term as shown by Wang et al. (2012).

Though Sewage Sludge and Wood Waste Fresh UEBCs at 6% and 10% application rates supplied significantly more urine-N than Sewage Sludge and Wood Waste CBS or Stored UEBCs, their application did not lead to higher N uptake in plant tissue. It is possible that urea molecules in fresh urine got trapped in small pores in Sewage Sludge and Wood Waste biochars and were slower to diffuse into soil water along a plant root gradient (Rasse et al., 2022). The comparatively higher temperature of these biochars points to a potentially higher porosity, particularly through the formation of micropores (Tomczyk et al., 2020). If urea-biochar did desorb from UEBCs in our study, it would likely be microbially available. Urea hydrolysis in soils is well documented as a rapid, first order reaction mediated by urea concentration and facilitated by common soil microorganisms utilizing the enzyme urease (Chin and Kroontje, 1963). Additionally, biochar application to soils is found to increase the ureolytic microbial abundance and urea hydrolysis rate (Liu et al., 2021). The linear relationship between Fresh urine-N applied and N uptake in plant tissue in urine-only treatments also supports this theory. Even at the high urine-N application rates of 6% and 10% for Wood Waste Fresh and 6% Sewage Sludge Fresh UEBCs, a potential soil priming effect is still evident. However, for Sewage Sludge-Fresh 10%, 4.02 mg urine-N were applied pot⁻¹, but 3.65 mg-N was present in the plant tissue. This implies a deleterious effect, potentially due to the high N content and/or high ash content of the Sewage Sludge biochar (Table 1). Future work should explore high application rates of UEBC prepared with feces-derived biochar in situ to determine if it is a suitable biochar for UEBC fertilization.

Figure 4 shows the total biomass response across treatments. Above-and-belowground biomass response was similar within treatments (Figures S4 and S5). Thus, all ANOVAs used total biomass as a response variable. Results from the three-way ANOVA show a significant difference between means (P < 0.001, three-way ANOVA, total biomass ∼ urine*biochar*application rate, Table 3). However, few treatments differed significantly when looking at Tukey HSD pairwise comparisons (Table 3). Total biomass in Walnut Shell-Stored 1%, Walnut Shell-Stored 10%, Walnut Shell-Fresh 6%, Sewage Sludge-CBS 1%, and Sewage Sludge-Fresh 10% were significantly lower than Stored 10% (P < 0.05, Tukey HSD, Table 3). Plant biomass in Walnut Shell-Stored 1% and Sewage Sludge-Fresh 10% were also significantly lower than Wood Waste-CBS 10% (P < 0.05, Tukey HSD, Table 3). Total biomass did not differ significantly between fertilized and unfertilized controls (Table 3). However, the unfertilized control was lower (0.251 ± 0.0327 g_dry) than the NPK fertilized control (0.275 ± 0.0245 g_dry). The organic fertilized control averaged 0.233 ± 0.0253 g_dry, likely due to a slow release of organically bound nutrients. The lack of a robust total biomass response overall may be due to hyphal nutrient foraging by the mycorrhizal inoculant in the soilless substrate (Cavagnaro et al., 2006). Future work should investigate the effect of UEBC on plant growth with and without mycorrhizal inoculant. Figures for leaf area of largest leaf, leaf number >1 cm, and height are available in the supplemental material as these growth indicators generally followed the same trend as total biomass (Figures S6–S8). No significant differences were found across treatments for final percentage of plants germinated (Figure S9).

However, some trends in the plant biomass response are evident when grouping predictor variables (biochar, urine, and application rate) in one-way and two-way ANOVAs (Tables S1–S7). No significant differences were found for total biomass between urine types across biochar types and application rates (P = 0.494, one-way ANOVA, total biomass ∼ urine, Table S1). This may be because the urine types demonstrate similar plant availability in the initial phases of plant growth when not adsorbed to biochar. While these data do not indicate the plant availability of the urine types themselves, we can conclude that one type is not significantly detrimental to plant growth over others. We did see a loss of more than 1,000 mg-N L⁻¹ from CBS urine compared to Stored urine. CBS urine averaged 3,211 ± 616 mg-N L⁻¹, while Stored urine averaged 4,241 ± 340 mg-N L⁻¹. Though we hypothesized that reduced CBS N content would lead to lower yield, this is still a substantial reduction in the potential fertilization quality of CBS urine and should be addressed in CBS systems. Closing individual urine containers between each use is unrealistic, though urine might be collated in larger, closed containers for decentralized urine-nutrient recovery to mitigate N losses. Another solution could be prevention of urease activity in the urine prior to nutrient recovery through acidification or alkalinization (Senecal and Vinnerås, 2017). Fresh urine averaged 3,619 ± 312 mg-N/L, with 3,355 ± 312 mg-urea L⁻¹, and 263 ± 28.7 mg- NH₄⁺ L⁻¹. The discrepancy between Fresh and Stored total N content may be due to incomplete activity of urease or experimental error.

A significant difference was found for total biomass between biochar types across urine types and application rates (P = 0.00445, one-way ANOVA, total biomass ∼ biochar, Table S2). Post hoc pairwise Tukey HSD tests showed that Walnut Shell differed significantly from urine-only treatments across biochar types and application rates (P < 0.001, Tukey HSD, Table S2). Average total biomass for Walnut Shell UEBCs was 0.248 ± 0.05 g_dry, while the average for urine-only treatments was higher at 0.278 ± 0.05 g_dry. This is likely due to more immediately available nutrients from urine-only treatments. No other significant differences were found between other biochar types or fertilized or unfertilized controls. There was no significant effect of application rate on total biomass response across urine and biochar types (one-way ANOVA, total biomass ∼ application rate, P = 0.205, Table S3). Regardless, optimal application rates likely depend on biochar and urine type in situ and should be optimized for different CBS settings based on urine conditions and treatment options, as well as biochar feedstock availability.

A significant difference between biochar/no biochar groups was found (P = 0.004, Wilcoxon Rank Sum test, total biomass ∼ biochar/no biochar, Table S4), with total biomass higher in urine-only treatments. This suggests that in the early growth stages, urine nutrients adsorbed to biochar may be less bioavailable than urine-only nutrients applied alone. These data did not support our hypothesis that UEBC treatments would outperform urine-only treatments. However, there are tradeoffs to applying urine as-is. As previously mentioned, application of urine alone can lead to leaching or gaseous losses of N, like synthetic N fertilizers (Wachendorf et al., 2005). Future research should investigate the best way to use urine as a fertilizer over the course of plant growth. It is possible some urine should be applied as a pre-plant application or sidedress application when using UEBC as a slow-release fertilizer.

Our research explores different UEBC preparations that could be potentially applied in CBS or other urine-diverting systems. We show that urine storage conditions have consequences for N retention in urine and subsequent adsorption to biochar, that biochar particle size is significant for urine-N sorption, and that urine-N bound to biochar likely releases more slowly than urine-N applied alone. However, future research should address additional questions on the actual application of UEBC in CBS and other urine-diverting systems. We chose a UEBC mixing ratio of 200 g:1 L based on prior experimentation that found this ratio favorable for urine-N adsorption. However, we recognize that this may be an unrealistic quantity of biochar to supply at the toilet or neighborhood scale. Future work should focus on creating replicable adsorption isotherms using real, urea-hydrolyzed, urease-inhibited, or CBS-style urine and various types of biochar. This is critical to create UEBCs with a predictable N content, which is ultimately necessary for their application and/or sale in real agronomic contexts. The economic feasibility of UEBC production in CBS systems should also be explored.

Additionally, more in situ data is necessary to investigate the potential of UEBC. Longer, field-scale studies should be implemented with different UEBC preparations and application rates. To fully understand the environmental impact of urine-based fertilizers, GHG emissions, NH₃ emissions, and N leaching from both UEBC production and application, as well as urine-only application, should be investigated across agronomic contexts. UEBC application studies that explore plant stress, such as water limitation, should also be undertaken, as the effect of UEBC on soil water dynamics after application is unclear.

Urine is an abundant source of plant available nutrients that is underutilized in agricultural systems. This research shows that application of Fresh, Stored, or CBS urine alone was positively linearly correlated with plant-N uptake, implying the immediate plant availability of urine-N. Across biochars, the <500-µm biochar size fraction retained significantly more N than the >500-µm fraction. Fresh urine-N adsorption to biochar was significantly greater than Stored or CBS urine in the >500-µm size fraction of Wood Waste and Sewage Sludge biochars. This could be explained by a difference in adsorption mechanisms for urea and NH₃/NH₄⁺ in urine to high pyrolysis temperature biochars or differential adsorption mechanisms of NH₃/NH₄⁺ and urea in the >500-µm and <500-µm biochar size fractions. Plant-N was not positively linearly correlated with urine-N application for Sewage Sludge and Wood Waste UEBCs and was loosely linearly correlated for Walnut Shell UEBCs. This implies that urine-N adsorbed to biochar is not bio-available in the early stages of plant growth. Future research should investigate the adsorption mechanisms of N species in real urine to biochar. Isotope dilution or tracer studies to understand urea-biochar and NH₄⁺-biochar sorption, desorption, and plant-N uptake would also help better explain the phenomena found in this study. This research may help CBS organizations to optimize urine-nutrient recovery and reuse with biochar, as different biochar and urine combinations lead to different agronomic outcomes.

The following databases were generated:

Bischak, E, Ché Fusi, S, Jeliazovski, J, Beheshtian, K, Ryals, R. 2022. Urine-enriched biochar: Coupling sustainability in sanitation and agriculture [Data set]. Zenodo. DOI: https://doi.org/10.5281/zenodo.7058293.

The supplemental files for this article can be found as follows:

Figures S1–S9. Tables S1–S7. PDF

The authors thank Brendan Harrison, Alexia Cooper, Zachary Malone, and Melisa Quintana for help designing, conducting, and interpreting data from this experiment. They also thank the Rotary District 5220 and UC Davis 2020 Global Fellowship for Agricultural Development for supporting this work.

This work was funded by the District 5220 2021 Rotary Scholarship, UC Davis 2020 Global Fellowship for Agricultural Development, and UC Merced Department of Life and Environmental Sciences.

The authors declare no competing interests.

Contributed to manuscript writing: EB, SCF.

Contributed to data analysis and data interpretation: EB, SCF, RR.

Contributed to conception and design of the study: EB, JJ, RR.

Contributed to acquisition of data: EB, KB.

Revised the first draft of the manuscript: All authors.

How to cite this article: Bischak, E, Ché Fusi, S, Jeliazovski, J, Beheshtian, K, Ryals, R. 2024. Urine-enriched biochar: Coupling sustainability in sanitation and agriculture. Elementa: Science of the Anthropocene 12(1). DOI: https://doi.org/10.1525/elementa.2022.00118

Domain Editor-in-Chief: Alastair Iles, University of California Berkeley, Berkeley, CA, USA

Knowledge Domain: Sustainability Transitions