Flue-gas desulfurization gypsum effects on urea-degrading bacteria and ammonia volatilization from broiler litter

Flue-gas desulfurization gypsum effects on urea-degrading bacteria and ammonia volatilization from broiler litter Christopher D. Burt,∗,1 Miguel L. Cabrera,∗ Michael J. Rothrock, Jr,† and D. E. Kissel ∗

Dep. Crop and Soil Sciences, Univ. of Georgia, 3111 Miller Plant Sciences Bldg., Athens, GA 30605; and † USDA-ARS U.S. National Poultry Research Center, 950 College Station Rd., Athens, GA 30605 mineralization in several 21-d experiments. The addition of FGDG to broiler litter increased EC by 24 to 33% (P < 0.0001), decreased urea-degrading bacteria by 48 to 57% (P = 0.0001) and increased N mineralization by 10 to 11% (P = 0.0001) as compared to litters not amended with FGDG. Furthermore, the addition of FGDG to broiler litter decreased NH3 volatilization by 18 to 28% (P < 0.0001), potentially resulting from the significantly lower litter pH values compared to unamended litter (P < 0.0001). Findings of this study indicate that amending broiler litter with 20% FGDG can decrease NH3 volatilization and increase the fertlizer value of broiler litter.

ABSTRACT A major concern of the broiler industry is the volatilization of ammonia (NH3 ) from the mixture of bedding material and broiler excretion that covers the floor of broiler houses. Gypsum has been proposed as a litter amendment to reduce NH3 volatilization, but reports of NH3 abatement vary among studies and the mechanism responsible for decreasing NH3 volatilization is not well understood. The goal of this study was to evaluate the effect of adding 20 or 40% flue-gas desulfurization gypsum (FGDG) to broiler litter on pH, electrical conductivity (EC), water potential, urea-degrading bacteria abundance, NH3 and carbon dioxide (CO2 ) evolution, and nitrogen (N)

Key words: broiler litter, ammonia, flue-gas desulfurization gypsum, nitrogen, urea-degrading bacteria 2017 Poultry Science 0:1–8 http://dx.doi.org/10.3382/ps/pex044

INTRODUCTION

soil acidity (Ap Simon et al., 1987; Galloway et al., 2002; Heald et al., 2012). The NH3 in broiler litter derives from the mineralization of organic N present in broiler excreta. Mineralization of organic N to NH3 is a microbially-mediated enzymatic reaction that requires enzymes produced by uric acid-degrading and urea-degrading populations (Rothrock et al., 2010). It is estimated that approximately 50 to 80% of the N in broiler litter can be converted to NH3 (Sims and Wolf, 1994; Ritz et al., 2004). To reduce NH3 volatilization in broiler houses, litter amendments such as acidifiers, chemical absorbents, and chemical/biological inhibitors have been investigated (Moore et al., 1996; Kithome et al., 1999; Cook et al., 2011; Loch et al., 2011; Timmons and HarterDennis, 2011). Gypsum (CaSO4 r2H2 O) has been proposed as a litter amendment to reduce NH3 volatilization, but results vary among studies and the mechanism responsible for decreasing NH3 volatilization is still not well understood (Wyatt and Goodman 1992; Sampaio et al., 1999; Oliveira et al., 2003, 2004; Loch et al., 2011). For example, Sampaio et al. (1999) found that the addition of gypsum to broiler litter reduced total bacterial counts and NH3 volatilization during a 49-d experiment. The authors cited a chemical reaction between gypsum and ammonium carbonate (Teuscher and Adler, 1965), as well as a reduction in bacterial counts

The United States poultry industry accounts for 28% (664,238 Mg) of the national livestock ammonia (NH3 ) emissions (USEPA, 2004). Specifically, broiler (Gallus gallus domesticus) production in the southeastern United States represents the largest segment of the poultry industry, with emissions representing 33 to 43% of the agricultural NH3 emissions in the region (Aneja et al., 2003; Stephen and Aneja, 2008). The majority of these emissions (55 to 82%) originate from broiler houses, with smaller amounts occurring during broiler litter storage and land application (Pain et al., 1998; Moore et al., 2011). The floor of the broiler production facilities is covered with broiler litter, which is a mixture of bedding material and excreta that is rich in organic and inorganic nitrogen (N). Ammonia volatilization from these N sources in the litter represents a major concern for producers because NH3 is considered the most harmful gas in broiler facilities (Carlile, 1984). Furthermore, the NH3 that is released to the environment by ventilation systems contributes to atmospheric particulate matter formation, acid rain deposition, and

 C 2017 Poultry Science Association Inc. Received May 12, 2016. Accepted February 2, 2017. 1 Corresponding author: [email protected]

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BURT ET AL. Table 1. Initial physiochemical characteristics of broiler litter used in laboratory experiments. Parameter Water Content Water Potential pH EC Total C Total N C/N Ratio

Unit

Value

g H2 O g−1 MPa −log[H+ ] μ S cm−1 ug g−1 ug g−1 —

0.63 −9.20 8.62 26.4 177,000 24,500 7.22

as possible mechanisms responsible for NH3 abatement in gypsum-amended litter. Oliveira et al. (2003) found that amending poultry litter with gypsum decreased litter pH and NH3 volatilization compared to unamended litter. Researchers hypothesized that the reduction in NH3 volatilization was due to gypsum’s high capacity to absorb moisture from litter, which reduced the activity of NH3 -producing bacteria. In contrast, a similar experiment was conducted by Oliveira et al. (2004) in which the quality of poultry litter treated with different amendments was investigated for three consecutive flocks. In that study, gypsum did not have a significant effect on litter pH or NH3 volatilization during the course of the study. Based on controversial results from previous experiments, additional studies are needed to investigate the effect of gypsum on microbial populations responsible for the production of ammoniacal N and subsequent NH3 volatilization. Gypsum produced by flue-gas desulfurization systems (FGDG) is an inexpensive source of gypsum that should be explored as a litter amendment by the broiler industry. Current information on the effect of adding FGDG to broiler litter on N transformations is very limited, and there is currently no known studies that investigate the effect of FGDG on urea-degrading bacteria abundance in broiler litter. Therefore, the goal of this study was to evaluate the effect of adding 20 or 40% FGDG to broiler litter on litter physiochemical parameters, urea-degrading bacteria (UDB) abundance, NH3 and carbon dioxide (CO2 ) evolution, and N mineralization in several 21-d experiments. It was hypothesized that FGDG amendments would reduce urea-degrading bacteria abundance, thereby decreasing N mineralization and subsequent NH3 losses.

MATERIALS AND METHODS Broiler Litter and Flue-Gas Desulfurization Gypsum (FGDG) Physiochemical descriptions of the broiler litters used in this study are shown in Table 1. Broiler litter samples were collected from a broiler house in Georgia, USA. The samples were passed through a 2-mm sieve and analyzed for total C and N, pH, electrical conductivity (EC), water potential, and water content. Total C and N in broiler litter was determined by dry combustion (Nelson and Sommers, 1982); pH was measured

using an AB150 pH meter by Fisher Scientific in a 1:5 (litter/deionized water) ratio, and EC was measured in a 1:5 (litter/deionized water) ratio using a CDM80 Conductivity Meter (Radiometer America, Cleveland, OH). Broiler litter water potential was measured using a WP4C Dewpoint Potentiameter (Decagon, Pullman, WA), and water content was determined by drying at 65◦ C for 48 hours. Flue-gas desulfurization gypsum was collected from a power plant in Illinois and analyzed for elemental composition using inductively coupled plasma optical emission spectrometry (ICP-OES) after acid digestion (USEPA, 1986). Flue-gas desulfurization gypsum used in all experiments contained 138,463 mg Ca kg−1 , 84,627 mg S kg−1 , 1,626 mg Fe kg−1 , 1,224 mg Mg kg−1 , 1,171 mg Al kg−1 , and less than 500 mg kg−1 of P, K, Si, and Na each.

Experiment 1: Effect of FGDG on Urea-degrading Bacteria and N Mineralization in Broiler Litter To determine the effect of FGDG on urea-degrading bacteria and N mineralization, broiler litter with two application rates of FGDG was incubated for 21 d. Treatments included: (i) broiler litter only (control), (ii) broiler litter + 20% FGDG, and (iii) broiler litter + 40% FGDG. There were six replicates of each treatment, all arranged in a completely randomized design. Each experimental unit consisted of 125 g of broiler litter (0.63 g H2 O g −1 ) placed in a 4-L glass container. Flue-gas desulfurization gypsum was added to designated experimental units based on broiler litter wet weight (20% and 40% FGDG treatments receiving 25 g and 50 g of FGDG, respectively), and the litter+FGDG mixtures were thoroughly mixed in each container. All containers were then closed and placed inside an incubator at 27◦ C for 21 d. Broiler litter from each experimental unit was sampled throughout the study. During each sampling event, a 2-g subsample of litter or litter+FGDG mixture was retrieved from each experimental unit to determine the water potential, and those subsamples were returned to the experimental units after analysis. The average water content for each treatment was then calculated using the average water potential for each treament and moisture release curves developed for each treatment (data not shown). Based on the estimated water content of each treatment, 5 g of broiler litter (dry weight) was retrieved from each experimental unit to be analyzed for pH, EC, urea, inorganic nitrogen, and ureadegrading bacteria. For FGDG treatments, 5 g of broiler litter (dry weight) was taken from each experimental unit by assuming that 20% and 40% of the dry material retrieved from the respective FGDG treatments represented the amount of FGDG in the mixture. Litter pH was measured in a 1:5 (litter/deionized water) ratio using an AB150 pH meter (Fisher Scientific), and it was also measured in 0.01 M CaCl2 on d 21 to account

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GYPSUM’S EFFECT ON BROILER LITTER

for any salt effect that the addition of gypsum may have on the pH probe. Urea was determined by shaking 1 g of litter with 100 mL of 1,000 mg L−1 AgSO4 for 5 min., and then filtering the solution through a 0.45μm hydrophilic polyethersulfone filter (Pall Life Sciences, Ann Arbor, Michigan). Extracts were analyzed following a colorimetric procedure (Keeney and Nelson, 1982). Ammonium-N and nitrate-N were determined by shaking 1 g of litter with 100 mL of 1 mol L−1 KCl for 45 min, and then filtering solution through a 0.45-μm filter. Extracts were analyzed following the colorimetric procedure described by Keeney and Nelson (1982). Concentrations of UDB were determined using a protocol similar to that of Kim and Patterson (2003). One gram of broiler litter was suspended in 30 mL of deionized water and shaken for 15 min. A 1-mL aliquot of poultry litter suspension was then pipetted into a dilution tube containing 9 mL of deionized water. A tenfold dilution was repeated twice. Aliquots from each dilution tube were plated on Christensen urea agar (Sigma-Aldrich, St. Louis, MI) following Miles and Mirsa (1938). After incubating the plates for 24 hours at 37◦ C, the colonies on each plate were quantified and results reported as UDB colony-forming units (CFU) per gram of dry litter.

Experiment 2: Effect of FGDG on Carbon Dioxide and NH3 Emmisions from Broiler Litter To determine the effect of FGDG on CO2 and NH3 volatilization, broiler litter with two application rates of FGDG was incubated for 21 d. Treatments, replications, and experimental design were the same as Experiment 1. Each experimental unit consisted of 10 g broiler litter (0.63 g H2 O g −1 ) placed in a 0.95-L glass container with a suspended vial containing 40 mL of 1 N H2 SO4 for trapping NH3 . Flue-gas desulfurization gypsum was added to designated experimental units based on broiler litter wet weight (20% and 40% FGDG treatments receiving 2 g and 4 g of FGDG, respectively), and each container was thoroughly mixed prior to incubation. All containers were then closed with screwcap lids fitted with rubber septa, and placed inside an incubator at 27◦ C for 21 d. CO2 concentration in the headspace and NH3 in the traps of each experimental unit were measured after 1, 2, 3, 4, 5, 9, 12, 15, 18, and 21 days. Triplicate 3-mL air samples for CO2 determination were collected with a syringe from the head space of each container and injected into a 2.0-mL glass sample vial. Concentrations of CO2 were determined using a Varian Star 3600 CX Gas Chromatograph with a Varian 8200 CX Auto Sampler (Varian Analytical Instruments, Sugarland, TX). After samples for CO2 determination were withdrawn from each experimental unit, containers were aerated and H2 SO4 traps were changed and analyzed for NH4 -N colorimetrically (Keeny and Nelson, 1982). The volume of air in the container was

calculated by subtracting the volume occupied by the litter from the air volume of an empty container and an NH3 trap. The rate of CO2 production from the samples was determined by subtracting background CO2 from the measured concentrations.

Experiment 3: Effect of FGDG on Nitrogen Balance A third 21-d incubation was conducted to calculate a N balance for litter amended with two rates of FGDG. Treatments in this experiment were the same as in the previous two experiments. There were four replications of each treatment, all arranged in a completely randomized design. Each experimental unit consisted of 16.3 g broiler litter (0.63 g H2 O g−1 ) placed in a 0.95-L glass container with a suspended vial containing 35 mL of 0.1 N H2 SO4 for trapping NH3 . The 20% and 40% FGDG treatments received 3.26 g and 6.52 g of FGDG, respectively, and the contents of each container were thoroughly mixed before incubating at 27◦ C for 21 d. H2 SO4 traps were changed after 1, 2, 3, 8, 12, 16, 19, and 21 days. After the H2 SO4 traps were removed on day 21, 500 mL of 1 M KCl was added to each experimental unit and shaken for 45 min in a reciprocating shaker set at 120 oscillations per minute. Each extract was then filtered through a 0.45-μm filter, and extracts were analyzed for NH4 + and NO3 − as previously described. NH3 from the H2 SO4 traps and NH4 + and NO3 − from the KCl extracts were calculated on a μg g−1 of dry litter basis and summed to calculate total nitrogen recovery.

Statistical Analysis Data were statistically analyzed using InfoStat v. 2012 (Di Rienzo et al., 2012). Before statistical analyses, all bacterial CFU concentrations were log10– transformed. Data from all the experiments were analyzed using a one-way analysis of variance as a completely randomized design. Statistical analysis of data for cumulative CO2 emissions and cumulative NH3 volatilization was performed using a repeated measures ANOVA test. Significant differences among treatment means were determined using Fisher’s protected LSD at P < 0.05.

RESULTS Experiment 1: Effect of FGDG on Urea-degrading Bacteria and N Mineralization in Broiler Litter Amending broiler litter with 20% and 40% FGDG significantly increased the EC (P < 0.0001) and water potential (P = 0.0001) after the first 24 hours, and this effect was sustained throughout the experiment (Table 2). The addition of FGDG to broiler litter significantly changed litter pH throughout the experiment.

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BURT ET AL. Table 2. Physiochemical characteristics of broiler litter (BL), broiler litter + 20% FGDG (BL + 20% FGDG), and broiler litter + 40% FGDG (BL + 40%) from Experiment 1 after 21 d of incubation at 27◦ C. pH Treatment BL BL + 20% FGDG2 BL + 40% FGDG2 P-value

EC3

Water potential

dH2 O

0.01 M CaCl2

μ s cm−1

MPa3

8.78 (0.02)1,a 8.33 (0.02)b 8.22 (0.02)c < 0.0001

8.28 (0.01)a 8.11 (0.01)c 8.19 (0.02)b < 0.0001

26.78 (0.25)c 33.36 (0.43)b 35.65 (0.15)a < 0.0001

− 10.29 (0.06)b − 8.58 (0.08)a − 8.57 (0.08)a < 0.0001

a-c Means within a column with different letters are significantly different according to Fisher’s LSD (P < 0.05). 1 FGDG addition was based on the litter wet weight. 2 EC = electrical conductivity, MPa = megapascal

After one d, the addition of 20% and 40% FGDG to broiler litter significantly decreased litter pH (8.19 and 8.17, respectively) compared to untreated litter (8.62) (P < 0.0001). At the end of the experiment, broiler litter receiving 40% FGDG had the lowest deionized water pH (8.22), followed by broiler litter + 20% FGDG (8.33) (Table 2). The pH of all experimental units was also measured in a 0.01 M CaCl2 solution on d 21 to dismiss any concerns regarding the effect of electrolyte concentration on pH measurements. The pH values measured in 0.01 M CaCl2 were lower than pH values measured in deionized water, but the pH of litter amended with FGDG was still significantly lower than the pH of unamended litter (P < 0.0001) (Table 2). Urea-degrading bacteria concentration at the beginning of the incubation was 1.33 ± 0.12 × 105 CFU g−1 litter and remained fairly constant in untreated litter throughout the incubation, ranging from 1.07 ± 0.23 × 105 to 1.72 ± 0.36 × 105 CFU g−1 litter (Figure 1A). The addition of 20% and 40% FGDG to broiler litter reduced the abundance of UDB on d 1 by 46% and 49%, respectively. Urea-degrading bacteria populations in FGDG-treated litters remained significantly lower than those in the untreated litter for the duration of the experiment (P = 0.0001) (Figure 1A). Concentrations of UDB in litter-amended with both rates of FGDG were similar for the first six d of the incubation (P = 0.42). On d 14 of the incubation, UDB concentrations in litter amended with 40% FGDG (4.37 ± 0.84 × 104 CFU g−1 litter) were significantly lower than concentrations in litter amended with 20% FGDG (7.15 ± 1.12 × 104 CFU g−1 litter) (P = 0.001), and this trend continued until the end of the incubation. After 21 d, amending broiler litter with 20% and 40% FGDG reduced UDB concentrations by 48% and 57%, respectively, compared to un-amended litter. The addition of 20% and 40% FGDG to broiler litter also significantly decreased urea concentrations throughout the experiment compared to un-amended litter (P < 0.05) (Figure 1B), and this resulted in significantly greater concentrations of NH4 + -N (P < 0.0002) (Figure 1C). Ammonium-N concentrations in FGDG-amended litter increased significantly on d 1 (P < 0.0001) (Figure 1C), and this trend continued for the duration of the experiment. After 21 d,

(a)

(b)

(c)

Figure 1. Urea-degrading bacteria, urea, and NH4 + -N concentrations in broiler litter (BL), broiler litter + 20% FGDG (BL + 20% FGDG) and broiler litter + 40% FGDG (BL + 40% FGDG) from Experiment 1 in which all treatments were incubated at 27◦ C for 21 d. (a) Urea-degrading bacteria (UDB) concentrations; (b) urea concentrations; (c) NH4 + -N concentrations. a-c On d 21, symbols with different letters are significantly different according to Fisher’s LSD (P < 0.05). 1 Symbols represent the mean of six replicates, and error bars represent standard error. 2 FGDG addition was based on litter wet weight.

broiler litter receiving 20% FGDG had the lowest concentration of urea (97.2 ± 10.9 μg urea g−1 litter), and the greatest concentration of NH4 + -N (15,849 ± 996 μg NH4 + -N g−1 litter) compared to litter receiving 40% FGDG and untreated litter.

Experiment 2: Effect of FGDG on Carbon Dioxide and NH3 Emmisions from Broiler Litter Amending broiler litter with 20% and 40% FGDG significantly reduced cumulative CO2 emissions after 21 d (P < 0.001) (Figure 2A). The majority of CO2 released from un-amended litter (58%) and FGDGamended litter (68-75%) occurred during the first

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GYPSUM’S EFFECT ON BROILER LITTER

with both rates of FGDG was similar throughout the incubation (P > 0.05), except on d 4 of the incubation (P = 0.014). Cumulative NH3 -N volatilization was significantly reduced by the addition of 20% or 40% FGDG during the course of the experiment (P < 0.0001) (Figure 2B). Approximately 47% of NH3 -N loss from un-amended litter occurred in the first two days of the experiment, while similar losses were delayed until d 3 for litter amended with FGDG. After 21 d, cumulative NH3 -N loss from un-amended litter represented 19.01% of the total N in the litter, whereas cumulative losses from poultry litter amended with 20% or 40% FGDG represented 14.7% and 15.3% of the total N, respectively (Figure 2B). Cumulative NH3 -N volatilization from litter amended with both rates of FGDG was similar throughout the incubation (P > 0.05), except on d one of the incubation (P = 0.02).

(a)

(b)

Experiment 3: Effect of FGDG on Nitrogen Balance Figure 2. Carbon dioxide (CO2 ) and NH3 volatilization of broiler litter (BL), broiler litter + 20% FGDG (BL + 20% FGDG), and broiler litter + 40% FGDG (BL + 40% FGDG) from Experiment 2 in which all treatments were incubated at 27◦ C for 21 d. (a) CO2 -C emissions; (b) cumulative NH3 loss. a-c On d 21, symbols with different letters are significantly different according to Fisher’s LSD (P < 0.05). 1 Symbols represent the mean of six replicates, and error bars represent standard error. 2 FGDG addition was based on litter wet weight.

5 d of the incubation. Cumulative CO2 emission from un-amended litter was significantly greater than emissions from litter amended with 20% and 40% FGDG on d 3 (P < 0.001), but there was no difference between FGDG treatments during this time (P = 0.487). On day d 5, there was not a significant difference in cumulative CO2 emission from any of the treatments (P = 0.195). However, on d 9, litter amended with 20% and 40% FGDG released significantly less cumulative CO2 (7,330 ± 432 μg CO2 -C g−1 litter and 7,156 ± 362 μg CO2 -C g−1 litter, respectively) than un-amended litter (8,560 ± 369 μg CO2 -C g−1 litter) (P < 0.001), and this trend continued for the duration of the incubation. Cumulative CO2 emissions from litter amended

Amending broiler litter with 20% or 40% FGDG resulted in about 40% more mineralized-N than in unamended litter after 21 d (P = 0.0001) (Table 3). The majority of mineralized-N recovered in all treatments was in the form of ammoniacal-N (92.6 to 95.2%) making NO3 − -N a small portion (4.8 to 7.3%) of the total N recovered. Cumulative NH3 -N loss from broiler litter was reduced throughout the experiment by the addition of 20% or 40% FGDG, and amending litter with 40% FGDG resulted in the least amount of cumulative NH3 -N loss after 21 d (8.43% total N). Concentrations of NH4 + -N were also affected by the addition of FGDG to broiler litter. Un-amended broiler litter contained approximately 50% less NH4 + -N than FGDG-amended litter, and the amount of NH4 + -N in litter increased with greater rates of FGDG application (Table 3). Even though there was a significant increase in NH4 + -N concentrations in litter amended with FGDG, NO3 − -N concentrations in FGDG-amended litter were not different from those found in un-amended litter on a weight basis (Table 3).

Table 3. Cumulative NH3 loss and inorganic nitrogen concentrations in broiler litter (BL), broiler litter + 20% FGDG (BL + 20% FGDG), and broiler litter + 40% FGDG (BL + 40% FGDG) from Experiment 3 in which all treatments were incubated at 27◦ C for 21 d. NH3 -N4 loss Treatment BL BL + 20% FGDG2 BL + 40% FGDG2 P-value

−1

μg g

2,854 (60)1,a 2,217 (10)b 2,061 (23)b < 0.0001

NH4 -N4 Litter −1

μg g

2,813 (50)c 5,870 (25)b 6,218 (27)a < 0.0001

NO3 -N4 Litter

Total N recovered

μ g g−1

μ g g−1

% Total N3

446 (5)a 450 (5)a 418 (15)a 0.072

6,117 (54)b 8,537 (32)a 8,698 (33)a < 0.0001

24.9 (0.22)b 34.8 (0.13)a 35.5 (0.13)a < 0.0001

a-c Means within a column with different letters are significantly different according to Fisher’s LSD (P < 0.05). 1 FGDG addition was based on the litter wet weight. 2 % Total N recovered = (NH3 –N loss + NH4 + -N + NO3 –N) / Total N. 3 NH3 -N = ammonia, NH4+ -N = ammonium-N, and NO3 –N = nitrate-N.

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BURT ET AL.

DISCUSSION Some authors have hypothesized that amending broiler litter with gypsum reduces NH3 volatilization due to a decrease in microbial activity associated with N mineralization (Sampaio et al., 1999; Oliveira et al., 2003; Mishra et al., 2013). In the present study, we documented that amending broiler litter with FGDG decreased the abundance of UDB by 48 to 57% after 21 d (Figure 1A), in agreement with findings by Sampaio et al. (1999) who observed a reduction in bacterial counts when litter was amended with different rates of gypsum plaster. The UDB reduction observed in this study coincided with a change in water potential and a significant increase in EC, potentially indicating that the addition of gypsum to broiler litter induced osmotic stress on urease-producing bacteria. Increases in osmotic stress encountered by microorganisms can cause cell dehydration and increased concentrations of solutes in the cytoplasm (Sleator and Hill, 2001), which can lead to a reduction in microbial biomass and respiration (Tripathi et al., 2006; Gennari et al., 2007; Chowdhury et al., 2011). Amending broiler litter with 20% and 40% FGDG decreased cumulative CO2 emsissions on d 3, but this effect on CO2 emissions diminished by d 5. After d 9, litter amended with both rates of FGDG released significantly less cumulative CO2 than un-amended litter, and this trend continued for the duration of the incubation. We suspect that the decrease in CO2 emissions from FGDG-amended litter measured in Experiment 2 (Figure 2A) is partially due to the reduction in culturable-UDB that was observed in experiment 1 (Figure 1A). In Experiments 1 and 3, amending broiler litter with both rates of FGDG resulted in significantly more plant-available nitrogen in the form of NH4 + -N than in untreated broiler litter after 21 d (P < 0.0001). Our hypothesis is that FGDG caused osmotic stress on UDB which caused these organisms to take in osmolytes (such as urea) to help counter the negative effects of dehydration and solute concentration. This, in turn would increase intracellular enzymatic urea hydrolysis which led to an increase in NH4 + -N. A related hypothesis is that cells that died as a result of the stress (evidence by the observed decrease in UDB) released intracellular enzymes that increased urea hydrolysis as well as mineralization of other organic molecules. Nitrate concentrations were very low in all studies, likely because the high NH4 + -N and high pH would indicate high NH3 concentrations, which would inhibit nitrifiers (Anthonisen et al 1976; Kim et al., 2006). Broiler litter is often sold as a N fertilizer for forage and row crops in the southeastern US, and our results indicate that the addition of FGDG increases the fertilizer value of broiler litter. The increase in N-mineralization that was observed in experiment three for FGDG-amended litter did not increase NH3 volatilization compared to un-amended litter (Table 3). In fact, the addition of FGDG to broiler litter decreased cumulative NH3 loss by 18 to 22% in

experiment two (Figure 2B) and by 22 to 28% in experiment three (Table 3). Cumulative NH3 losses from litter amended with 20% and 40% FGDG were similar in experiments two and three, indicating that the application of 40% gypsum used in Oliveira et al. (2003, 2004) is not needed to significantly reduce NH3 from broiler litter. The reduction in NH3 loss from FGDG-amended litter was potentially due to the decrease in litter pH that was observed in Experiment 1 (Table 2). Urea hydrolysis generally causes an increase in litter pH (Equation 1) (Kissel and Cabrera, 1988), and NH3 volatilization is known to increase as the pH increases to 8 or more (Reece et al., 1979). CO(NH2 )2 +2H2 O + H+ → NH4 + + HCO3 −

(1)

As the pH of broiler litter increases to 8.2 and above, HCO3 − deprotonates to form CO3 2− (Equation 2), and in the presence of Ca2+ , CO3 2− will precipitate as CaCO3 − (Equation 3). HCO3 − ↔ CO3 2− + H+

(2)

Ca+2 + CO3 2− → CaCO3

(3)

The precipitation of CaCO3 − in equation [3] favors the consumption of HCO3 2− leading to the release of additional H+ (equation [2]) that can buffer against increases in pH. Furthermore the deprotonation of HCO3 − , (equation [2]) and precipitation of CaCO3 (equation [3]) would reduce the amount of CO2 emitted, which is what was observed in Experiment 2 when broiler litter was amended with FGDG. An additional factor that could contribute to a reduction in CO2 emission is the reduction in UDB observed in Experiment 1 (Figure 1A). Although CaCO3 precipitation was not measured in this study, our measurements of pH and CO2 emissions indicate that it is likely that more CaCO3 precipitates in FGDG-amended litter than in un-amended litter, which in turn would buffer against pH increases and lead to lower levels of NH3 volatilization. Future studies that examine the effect of FGDG addition on litter characteristics should consider calculating the amount of CaCO3 that is produced in FGDG-amended litter compared to untreated litter. It should be noted that microbial carbonate precipitation generally occurs on the external surface of bacterial cells (Castanier et al., 1999), and the accumulation of CaCO3 on the cell surface limits exchange of materials with the environment (Silva-Castro et al., 2015). In addition, the expulsion of H+ from bacteria reduces the pH of the microenvironment surrounding the cell, increases intracellular pH, and reduces the proton pool available for biological processes (Norris et al., 1996). These reactions are detrimental to cell survival, and

GYPSUM’S EFFECT ON BROILER LITTER

could explain the reduction in the number of culturableUDB that was observed in FGDG-amended treatments (Figure 1A). In summary, our results indicate that the addition of FGDG at a rate of 20% or 40% of broiler litter weight altered the water potential of litter, while increasing EC as compared to the un-amended controls. These conditions were detrimental to the survival of UDB, and led to an increase in N mineralization possibly due to an increase in enzymatic activity. Lower pH in FGDG-amended litter resulted in less cumulative NH3 volatilization and greater concentrations of NH4 + -N in litter. While being significantly different than the unamended controls, the addition of 20% or 40% FGDG to broiler litter resulted in similar amounts of UDB, urea, cumulative CO2 and NH3 loss, and nitrogen mineralization after 21 d. Thus, amending broiler litter with 20% FGDG is an effective strategy to decrease NH3 volatilization and increase the fertilizer value of broiler litter.

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