Comparing biochar-swine manure mixture to conventional manure impact on soil nutrient availability and plant uptake – A greenhouse study

: The use of swine manure as a source of plant nutrients is one alternative to synthetic fertilizers. However, conventional manure application with >90% water and a low C:N ratio results in soil C loss to the atmosphere. Our hypothesis was to use biochar as a manure nutrient stabilizer that would slowly release nutrients to plants upon biochar-swine manure mixture application to soil. The objectives were to evaluate the impact of biochar-treated swine manure on soil total C, N, and plant-available macro and micronutrients in greenhouse-cultivated corn (Zea mays L.) and soybean (Glycine max (L.) Merr.). Neutral pH red oak (RO), highly alkaline autothermal corn stover (HAP), and mild acidic Fe-treated autothermal corn stover (HAPE) biomass were pyrolyzed to prepare biochars. Each biochar was surface-applied to swine manure at a 1:4 (biochar wt/manure wt) ratio to generate mixtures of manure and respective biochars (MRO, MHAP, and MHAPE). Conventional manure (M) control and manure-biochar mixtures were then applied to the soil at a recommended rate. Corn and soybean were grown under these controls and treatments (S, M, MRO, MHAP, and MHAPE) to evaluate the manure-biochar impact on soil quality, plant biomass yield, and nutrient uptake. Soil OM significantly (<0.05) increased in all manure-biochar treatments; however, no change in soil pH or N was observed under any treatment. No difference in soil ammonium between treatments was identified. There was a significant decrease in soil M3-P and soil NO 3 - for all manure-biochar treatments compared to the conventional M. However, the plant biomass nutrient concentrations were not significantly different from control manure. Moreover, an increasing trend of N and decreasing trend of P in the plant under all biochar-manure treatments than the controls were noted. This observation suggests that the presence of biochar is capable of influencing the soil N and P in such a way as not to lose those nutrients at the early growth stages of the plant. In general, no statistical difference in corn or soybean biomass yield and plant nutrient uptake for N, P, and K was observed. Interestingly, manure-biochar application to soil significantly diluted the M3-ex-tractable soil Cu and Zn concentrations. The results attribute that manure-biochar has the potential to be a better soil amendment than conventional manure application to the soil. the results.


Introduction
Corn (Zea mays L.) and soybeans (Glycine max (L.) Merr.) crop rotation is a common practice in the Midwest U.S. Typical Midwest crop rotations and high fertilizer application may increase yield. However, both corn-corn and corn-soybean rotational systems and high inorganic fertilizer application can negatively impact soil C sequestration [1]. Figure 1. Concept of sustainable animal and crop production system -the proposed role of biochar. (Stage 1) Surface-applied biochar to manure mitigates short-and long-term emissions of odor and gaseous pollutants, retaining more nutrients in the manure. (Stage 2) Manure-biochar mixture is used as a value-added fertilizer, improving soil organic matter, nutrient utilization by plants, and lowering nutrient runoff risk.
The research compares the soil physicochemical properties and plant-available nutrients with conventional manure and three biochar-manure mixtures under corn and soybean over two months. In this greenhouse study, we investigated biochar-manure treatments' impact on soil physicochemical properties (pH, bulk density, total C and N) and major plant nutrients (N, P, K), as well as M3-extractable minor nutrients under corn and soybean. The three biochar manure mixtures were prepared from neutral pH red oak (RO), highly alkaline autothermal corn stover (HAP), and mild acidic Fe-treated autothermal corn stover (HAPE) feedstocks followed by incubating the biochar with manure for a month and called MRO, MHAP, and MHAPE respectively. This research also addressed the impact of Fe-modified corn stover biochar application on manure to sorb nutrients followed by soil application as an amendment.

Soil collection, biochar, manure, and manure-biochar incubation
A well-drained Hanlon (coarse-loamy, mixed, superactive, mesic Cumulic Hapludolls) soil was collected in bulk from the Iowa State University Applied Science/Moore research farm in the fall of 2019, after soybean harvest. A corn-soybean rotation was in place for the previous 5 years, and no evidence of swine manure application was recorded for the last 20 years. A composite surface soil (0-10 cm) was collected using a shovel and stored in buckets with lids to reduce moisture loss at 4 °C for three months until the trial.
The swine manure was collected from Iowa Select Farms in fall 2019, and then it was stored in a bucket with a lid at laboratory temperature (23-24 °C). By weight, 1,000 g of manure and 250 g of biochar were mixed at a 4:1 ratio and incubated for four weeks under laboratory T (23-24 °C). A 1,000 g control manure sample was also incubated under the same condition for comparison. During incubation, all the containers were covered with a perforated aluminum foil to have air exchange without losing much moisture to the atmosphere. Then, the manure treatments were stored in airtight glass containers (keep the moisture constant) at 4 °C to reduce the microbial activity until applied to the soil in a month. These stored manure and manure-biochar mixtures were considered as treatments further and applied to the soil.
2.2 Soil preparation, greenhouse, and pot experiment A part of the total field moist soil was dried, sieved (<2mm), and stored to do the baseline soil analysis. The rest of the bulk soil sample was crushed by hand to break soil clods and remove roots, and other larger (>2 cm) debris from the soil. The soil was then stored in a bucket with a lid for the greenhouse pot study. Inside the greenhouse, a recommend daytime (16 h) temperature was set to 29 °C, and nighttime (8 h) temperature was set to 20 °C for the experiment's duration.
Each of the 40 generic plastic pots (10.0 cm inner diameter & 11.4 cm height) was filled with 0.5 kg of Hanlon soil. Twenty pots were used to grow corn and 20 pots for soybean. The pots were labeled with manure-biochar mixtures codes for treatments: M = manure control; S = soil control; MRO = manure+red oak biochar; MHAP = manure+ highly alkaline porous biochar; MHAPE = manure+highly alkaline porous engineered biochar (Table 1). There were four replicates of each treatment as given in the schematic (Figure 2). The pots were randomized within replication. The amount of manure treatment applied to the soil followed the 135 kg/ha (120 lb/ac) recommended rate for P. The decision on the amount of manure and manure-biochar mixture addition as a treatment was challenging but key factor as they had a different amount of macro and micro-nutrients at the end of the manure-biochar incubation. Specifically, two important factors are considered to decide on the amount of nutrient addition based on the P content of the mixture and reduce the variability and complexity among the biochar-manure mixtures. We considered (factor 1): the plant-available P content of organic fertilizer as one of the crucial plant nutrients, and (factor 2) the greater affinity of Fe minerals for P in the HAPE biochar [16] than RO or HAP feedstock biochars. After the treatment application, 50 mL of water was added every other day for a week. After equilibrating the pots for a week, three corn (Pioneer P1197AMXT) and three soybean (Pioneer P31A22X) seeds were planted into the respective pot and watered every other day.

Soil and biomass analysis
The greenhouse experiment was conducted for eight weeks. All the corn and soybean pots were observed for plant germination, and the plant growth stage was determined using the leaf collar method for corn [38]. Soybean maturity was determined using the method described by Fehr et al. (1971) [39]. At the end of 8 weeks, the soil was separated from the plant roots. Then the soil was dried, sieved (<2 mm), and sent to a service laboratory for analysis of pH, OM, total C, total N, Mehlich3 (M3) extractable soil elements, and 2M KCl extractable nitrate-N, ammonium-N using methods referred to in section 2.1. All soil sample's pH was analyzed in water (1:1 soil:water) using the glass-electrode meter.
Plant biomass was collected by cutting each plant from the pot at 2-3 cm above the soil surface and placed in a labeled bag. These bags were dried for a week in an oven at 65 °C. After a week, the plant biomass with a constant weight was considered as the dry biomass yield. The biomass samples were used to determine the plant nutrient concentration, and when combined with dry biomass yield, calculated plant major element N, P, and K and other nutrients uptake. Total N and C for plants were analyzed by combustion method using C/N analyzer as described by Method P2.02 [37]. Plant minerals were extracted by microwave digestion of the biomass with concentrated nitric acid and hydrogen peroxide in a closed Teflon vessel and then analyzed with ICP-OES as described by Method P-4.30 [37].

Statistical Analysis
The statistical analysis was completed using R. The experiment has three biochar manure mixtures, one manure control, and one soil control, with four replicates of each treatment and two types of plants (corn and soybean), for a total of 40 pots. The treatment effects on soil nitrate, ammonium, P, K, all Mehlich3 extractable elements, biomass yield, and nutrient uptakes were the response variable for ANOVA, and the least significant difference was computed using Tukey's adjustment. A p-value <0.05 was considered statistically significant.

Results
The soil used in this study had a neutral pH (7.6), containing 2.84% of organic matter, 1.88% of total C, and 0.17% of total N. After incubation of the manure-biochar mixture, pH ranged for the mixtures from neutral to alkaline (7.5-9.9), MRO being the highest and MHAPE in the lowest in the range. The control manure (M) was alkaline (9.2), with 37.4% TC and 18.1 % TN. The fast pyrolysis red-oak (TC 78.5% and TN 0.6), corn stover (TC 61.4 and TN 1.2%), and Fe-engineered corn stover (TC 36.4 and TN 1.2) biochars had a high total C:N range (30-130); however, incubation of the biochar with manure decreased the total C:N to 13-52 range. Details of the manure, biochar, manure-biochar mixture and their characteristics are available in   [20] .
The amount of manure or manure-biochar mixture added end-up as significantly different amount of nutrient elements addition [20] to corn and soybean pots ( Table 2). The nutrient amount was calculated (by weight in g) from the manure and manure-biochar mixtures' nutrient value after the 1-month incubation at laboratory T. Treatments end up with approximately 6.2 g of manure, 3.6 g of MRO, 9.8 g of MHAP, and 6.5 g of MHAPE. The total N added was the lowest in the MRO treatment. The manure-biochar treatments applied had more macro and micronutrients than manure (M) control except for Cu and Zn. Table 2. Comparison of the mass of elements by different treatment added (before planting) by the manure and manure-biochar application. The mean was calculated and compared for sample size n=4. Different letters signify statistical differences between treatments at p<0.05 (column-wise).

Impact of treatments on corn planted soil
Manure (M) application to the soil significantly increased (p< 0.05) the soil NO3concentrations ( Figure 3A) relative to the control soil (S) or any of the manure-biochar mixture (MRO, MHAP, MHAPE) treated corn-planted soil. An increasing (numerically) trend in soil NH4 + was observed for all manure-biochar treatments (MRO-NH4 + : 5.2; MHAP-NH4 + : 6.0; MHAPE-NH4 + : 6.7) compared to manure (M-NH4 + : 5.0) or soil control (S-NH4 + : 6.6) soil. However, the increase was not significantly different from the controls due to the high variability of soil NH4 + concentrations between replicates of manure-biochar treated soils ( Figure 3B). A significant (p<0.05) increase in Mehlich3 extractable P was observed for the manure (M) applied soils compared to the manure-biochar mixture (MRO, MHAP, MHAPE) treated soil pots ( Figure 4A). Generally, a significant (p<0.05) increase in soil K was also observed for all manure and manure-biochar mixture treatments compared to soil control (S). However, there was no significant K increase between manure and manure-biochar treatments ( Figure 4B).  The addition of the manure (M) or manure-biochar (MRO, MHAP, MHAPE) mixtures to soil did not show a significant increase in the soil pH in pots with corn ( Table 3). The addition of the manure-biochar mixtures to soil influenced the percent OM and total C compared to manure. To be specific, MHAP and MHAPE treatments significantly increased the soil OM (%) compared to manure (M) or soil (S) control. The manure-biochar treatments did not increase the soil total-N. The total C:N ratio was significantly higher in the biochar-manure samples compared to either manure or soil controls.
No significant difference between soil Ca or Mn concentrations was found between treatments; however, MRO and MHAP treatments showed a significant decrease in the soil Mg concentration. The biomass of HAPE biochar was pretreated with Fe, so a significant increase in soil Fe for MHAPE treatment was evident. The addition of manure to soil significantly (p<0.001) increased the soil Cu and Zn. Manure-biochar mixture application to soil had a significant decrease in soil Cu and Zn concentrations compared to the manure-treated soil pots (Table 3).   Figure 5 shows that soils that grew soybeans had relatively higher NO3 -8 concentrations for manure (M) and soil (S) control than manure-biochar mix- 9 ture treated soil (Fig. 5A); however, the difference was not significant for both 10 NO3 -& NH4 + due to high variations among the replicates (Fig. 5). treated soils had significantly lower M3-P concentration than manure treat- 22 ment. The soils treated with MHAP had the lowest P; however, no significant 23 differences among treatments were observed. 24 The MHAPE treated soil had significantly higher K among all treat-25 ments ( Figure 6B). Control soil (S) had the lowest K, while MRO and MHAP 26 were not significantly different from the manure (M) treated pots. Total C for manure-biochar treated soybean soils were higher, and 35 MHAP and MHAPE were significantly higher than the controls ( Table 4). The 36 trend of total N increased by the treatments; however, treatments were not 37 statistically different. Additionally, the total C:N ratio also stayed higher for 38 all manure-biochar treatments than manure or soil control. None of the ma- nure-biochar treatments increased or decreased the soil pH significantly, alt- 40 hough the manure or the manure-biochar mixtures were either alkaline or 41 acidic before application soil [20]. 42 Only MHAPE treatment showed a significant increase in soil nutrients 43 such as Ca, Mg, Fe, and Mn after the eight weeks of greenhouse soybean 44 growth experiment. An obvious increase in Fe concentration for MHAPE 45 could be attributed to the pretreatment of biomass with Fe to specifically sorb 46 negatively charged ions from the system upon its application. The addition of manure significantly increased soil Cu and Zn concentrations (Table 4). 48 However, the Zn concentration dropped for the biochar manure treatments 49 significantly compared to manure control. A low Cu concentration trend was 50 also observed for all manure-biochar treatments compared with controls.  gence. All the other soybean pots showed the healthy growth of the plants. 69 No significant change in biomass yield (Figure 7) was observed between 70 treatments. The total nutrient uptake data (

Discussion
Pots of corn and soybean under different treatments received biocharmanure mixture based on the mixture P concentrations. Soil total C and OM were slightly different under corn and soybean (Tables 3 & 4). The addition of biochar-manure mixtures (instead of conventional manure) increased soil total C and OM for both plants. However, only the OM under corn soil and total C increased under soybean and were significant. Long-term application of liquid swine manure to the soil can also accelerate the native soil C mineralization and is followed by its loss to the environment as CO2 [40]. The recalcitrant nature of biochar is capable of inhibiting soil C loss, and improved soil OM was reported in earlier studies [9,12]. After incubation under laboratory condition % TC of control manure was 38.2 %, and MHAPE had 36.3 % TC [20]. The soybean and corn pots received approximately 6.2 g manure and 6.5 g MHAPE for the control and MHAPE treatment, respectively. Interestingly, pots of both plants ended up with high TC and a significantly high amount of OM for MHAPE than control manure pots, which can be attributed to the fact that manure C and soil C were stabilized by biochar. This supports the earlier findings that biochar-soil interaction stimulates the pyrogenic C mineralization; besides, the recalcitrant nature of the biochar C improves OM sorption's process to biochar and physical protection of the labile C [11]. The wood biochar-manure mixture (MRO) had 50.2% of TC. Thus the MRO treatment has also increased the soil total C and OM. However, the increase was not significant in comparison to the manure control (M). This observation also speculates that biochar from different feedstocks and manure interaction is a complex phenomenon.
Manure or biochar-manure treatments did not impact the soil pH significantly for either soybean or corn. Several earlier studies have reported that biochar's application increases the pH of soils [7,14]. In contrast, this study reports biochar influences nutrient availability without significantly changing soil pH. Pots receiving MHAPE treatment had a slightly lower pH than any other treatments or soil control. The FeSO4 pretreatment influencing the relatively low pH of the MHAPE material could be the reason behind this observation. Irrespective of the lower pH of the MHAPE, the treatment did not change the overall soil pH and has significantly influenced most soil nutrients availability than other manure-biochar or control treatments under the plants studied here.
The prevalent form of inorganic form of N present in the manure is NH4 + [41]. Upon application of manure to the soil, NH4 + mineralized to NO3quickly, and thus, a conventional manure application could increase NO3leaching loss from soil [8]. However, our findings support the speculation by Laird et al., 2010a that the presence of biochar can hinder the NH4 + to NO3mineralization by sorbing the NH4 + present in the manure and inhibit the nitrification process (Figures 3 and 5). A low comparable ammonium concentration under all treatments and high NO3for manure treatments indicates a possible slow NH4 + to NO3mineralization. Our previous short experiment [20] reported a significant increase in NO3and NH4 + under MHAPE biochar treatment than manure, but no such pattern was observed in this experiment. This study observed a higher concentration of NO3under manure (M) control than biochar-manure mixture treatment; however, the values were highly variable to differentiate treatments under soybean significantly. The presence of plants might also have influenced the soil inorganic N dynamics, making the observation complex.
Application of MHAPE to the soil under corn and soybean had significantly boosted the M3-Fe, and this observation is undoubtedly related to biomass pretreatment with Fe (Tables 2, 3, and 4). The availability of Mn was also significantly increased for MHAPE treated soybean pots but not corn among all treatments. Biochar-manure treatment promoting the soil Mn availability is also reported by Lentz and Ippolito (2012) [12], suggesting a synergistic effect of the treatment on soil and microbial activity. The MHAPE treatment resulted in significantly higher concentrations of soil Ca and Mg than manure (M) treatments for the soybean pots, but no such significant increase in those elements was observed under corn-grown soils. In contrast, MRO and MHAP decreased the Mg concentrations under corn but did not impact the soybean soils. Corn individual nutrient uptake is more than individual soybean nutrient uptake of N, P, and K as reported by Lv et al., 2014 [42]. Besides, the unique plant uptake of the nutrients at the earlier stage of the crop growth could impact the soil nutrient availability differently than a mature plant. Different soil Ca and Mg patterns for corn and soybean under manure-biochar treatments suggest that high concentrations of these elements in the soil make their availability unpredictable. The concentration of Mg in corn receiving MHAP and MRO treatments was significantly high in comparison to other treatments, whereas in the case of soybean, both Ca and Mg were significantly higher in soil under only MHAPE treatment. An increase in soil Mg but not Ca for the Fe-pretreated biochar-manure was reported by   [20].
Manure application to soil significantly increased M3-P under both corn and soybean; besides, no biochar manure mixture treatments significantly increased the M3-P. A relatively low M3-P for MHAPE treatment among all biochar manure treatments under corn may have resulted from specific adsorption of soil-P onto the biochar Fe surface, making the P less available in the soil [16]. Allen and Mallarino (2008) [5] reported that multiple manure application in a conventional way overloads the soil P; however, the application of manure-biochar mixture in this research showed that it could lower plant-available P loss from soil and resolve manure management issues.
Manure application to the soil as a fertilizer may result in the accumulation of Cu and Zn [8,12,20]. In general, the Cu and Zn concentrations were consistently lower (<0.05) under all the manure-biochar treatments compared with the manure control under both corn and soybean. In contrast to the findings of Lentz and Ippolito (2012) [12], our study reports that biochar-manure mixture may improve soil Cu and Zn concentrations elevated by the manure application. Manure, a source of Cu and Zn, when incubated with biochar (Table 2), diluted their mass in the mixture [20]. The presence of biochar can reduce the concentration of soil Zn of manure-treated soil, as was reported in a previous study [8].
Manure as fertilizer can improve biomass yield. In this study, there was an increasing biomass yield trend for both corn and soybean receiving manure treatments than biochar-manure treatments (except for corn-MRO); however, changes were not significantly different. Because of the plant's early termination, the biomass yield values were low and made the biomass yield interpretation challenging. A decreasing trend of Mn uptake by corn with increasing biochar application rate was also reported by Rogovska et al., 2014, besides the Mn uptake was found to be within the sufficiency range for plant growth [10]. Similarly, a lower Mn uptake was also observed for the current study; however, the concentration was within the sufficiency range. Additionally, the variability and biochar-manure mixture application had no significant difference in plant nutrient uptake than manure or soil treatments. We believe this short study has identified that all plants were healthy under both manure and manure biochar treatments. A long-term field-based trial is warranted to determine the long-term effect of manure-biochar on the soilplant environment.

Conclusions
An increase in soil OM and total C under all treatments suggests the biochar-manure mixture has the potential to improve soil C sequestration compared to a conventional manure application to soil. Biochar incubation with manure stabilizes manure P and releases an optimum amount of plant-available P at the early plant growth stage. Moreover, the high NO3-concentration under conventional manure treatment than manure-biochar treatments suggests that the presence of biochar with manure may reduce the risk of N (as NO3-) leaching loss to the environment. This could save the soil N and P for a later time when the plants need it and lower the risk of N or P deficiency. These observations support our working hypothesis that the use of biochar is a manure nutrient stabilizer that, upon soil application, slowly releases nutrients to plants. Overall, no particular pattern was observed for the soil nutrients availability for any biochar-manure treatment under corn and soybean. However, biochar diluted Cu and Zn present in the manure and thus reduced the risk of their accumulation or release to the soil systems. This study also showed that biochar-manure mixture application to soil did not hinder plant nutrient uptake during this two-month greenhouse experiment. The results suggest that manure-biochar could be a better soil amendment than conventional manure application to the soil. A long-term field-based trial is warranted to determine the long-term effect of manure-biochar on the soil-plant environment.

Supplementary
Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: title. All corn plants displayed; soil trial row is closest to the camera.