d’éthanol dans l’Est du Canada
Response to nitrogen of sweet pearl millet and sweet sorghum
grown for ethanol in eastern Canada
Marie-Noëlle Thivierge, Martin H. Chantigny, Gilles Bélanger, Philippe Seguin,
Annick Bertrand, and Anne Vanasse*
M.-N. Thivierge and A. Vanasse, Dép. de phytologie, Univ. Laval, 2425 rue de l'Agriculture, Québec, Canada, G1V 0A6; M.H. Chantigny, G. Bélanger, and A. Bertrand, Agriculture and Agri-Food Canada, 2560 Hochelaga Blvd., Québec, Canada, G1V 2J3; P. Seguin, Macdonald Campus, McGill
University, 21111 Lakeshore Road, Ste-Anne, Québec, Canada, H9X 3V9. *Corresponding author (Anne.Vanasse@fsaa.ulaval.ca)
35
Résumé
Le millet perlé sucré et le sorgho sucré sont deux cultures envisagées pour la production d’éthanol dans l’est du Canada, mais leur réponse à l’azote (N) n’est pas bien connue. Les deux espèces ont été cultivées à deux sites situés au Québec, et fertilisées avec du nitrate d’ammonium (0 à 160 kg N ha-1) et des lisiers de porc et de bovin (80 kg N total ha-1). Les meilleurs rendements en sucres solubles ont été obtenus avec 86 à 91 kg N ha-1, et ceux en matière sèche avec 107 à 121 kg N ha-1. Pour une même dose de 80 kg N total ha-1, l’efficacité fertilisante des lisiers a varié de 15 à 52 % de celle de l’engrais minéral. La concentration en sucres solubles du sorgho sucré était supérieure de 68% à celle du millet perlé sucré. Le sorgho sucré semble donc plus prometteur pour la production d’éthanol.
36
Abstract
Sweet pearl millet [Pennisetum glaucum (L.) R.BR.] and sweet sorghum [Sorghum bicolor (L.) Moench] are considered for ethanol production in eastern Canada but their response to N fertilization is not well established. Our objectives were (i) to compare both species for dry matter (DM), water- soluble carbohydrate (WSC), and estimated ethanol yields, and (ii) to determine their response to mineral N rate (0, 40, 80, 120, 160 kg N ha-1), (iii) mineral vs. organic N (liquid swine and dairy manures at 80 kg total N ha-1), and (iv) single vs. split N application (80 kg ha-1 mineral N). The two species were grown for two years on sandy loams in two ecozones (Mixedwood Plains [MWP] and Boreal Shield [BS]) with contrasting temperature regime. Both species responded similarly to mineral N fertilization, with maximum WSC yield at 86 kg N ha-1 at MWP and 91 kg N ha-1 at BS, and maximum DM yield at 121 kg N ha-1 at MWP and 107 kg N ha-1 at BS. Mineral N fertilization at 80 kg ha-1 resulted in higher DM and WSC yields than the manures, which showed fertilizer N equivalences varying from 15 to 52%. Splitting mineral N rate between seeding and the 4-leaf stage did not improve yield compared to a single application. Sweet sorghum had higher averaged WSC concentration and yield than sweet pearl millet (249 vs. 134 g WSC kg-1 DM; 3.41 vs. 2.02 Mg WSC ha-1) and appears more promising for ethanol production in eastern Canada.
37
Introduction
The search for alternative energy sources is a major concern both scientifically and politically, mainly because of worldwide increases in greenhouse gas emissions, concerns about availability, efficiency and safety of crude oil extraction, and need for energy self-sufficiency (Prasad et al., 2007; Yuan et al., 2008; De Vries et al., 2010; Sawargaonkar et al., 2013). Biofuels from plants, the combustion of which returns to the atmosphere C formerly fixed by photosynthesis, are widely used in North America. Canada, the United States, and Mexico have regulations to blend ethanol into the national gasoline pool (NEB, 2013).
Corn (Zea Mays L.) starch is the sole feedstock used in eastern Canada for ethanol production at an industrial scale. Corn necessitates high amounts of fertilizer, especially N, to produce high grain yields. Manufacturing, transport and use of N-based fertilizers constitute the main energy-consuming process in crop production (Amaducci et al., 2004; AGECO, 2006; Piringer and Steinberg, 2006; Tamang et al., 2011). Nitrogen is also one of the main pollutants from agriculture, escaping the soil in many ways after application (Janzen et al., 2006; De Vries et al., 2010). Another concern is that biofuels relying on starch or sugar from traditional food crops induce a stress on food commodities (Gomez et al., 2008). There is therefore a challenge to fulfill both food and energy needs, and to ensure that biomass production generates the smallest impact on the environment (Boehmel et al., 2008; Sawargaonkar et al., 2013).
Sweet pearl millet [Pennisetum glaucum (L.) R.BR.] and sweet sorghum [Sorghum bicolor (L.) Moench] are among the species that could possibly be used for ethanol production in eastern Canada because they accumulate more WSC in their stalks than conventional cultivars of the same species. Indeed, a sugar-rich juice can be extracted from the stalks and directly fermented into ethanol, whereas the pressed stalk residues (bagasse) can be used as silage for cattle, therefore contributing to feed and non-food production (Wang and Liu, 2009; Sawargaonkar et al., 2013). The potential of sweet pearl millet for energy production has recently been investigated in eastern Canada (Bouchard et al., 2011; Leblanc et al., 2012; Crépeau et al., 2013; Dos Passos Bernardes et al., 2014). Dry matter yield of 20.4 Mg ha-1 (Dos Passos Bernardes et al., 2014) and WSC concentration of 143 g kg DM-1 (Leblanc et al., 2012) were reported. Leblanc et al. (2012) observed that DM yield of sweet pearl millet increased with N rates while WSC concentration was not affected, resulting in an
38
increase in WSC yield with N rates. Using a linear-plateau function, Leblanc et al. (2012) determined that maximum WSC yield was reached at 90 kg N ha-1.
Sweet sorghum was suggested for ethanol production in the early 80’s (Kresovich and Henderlong, 1984) mainly because of its high content in WSC, its rapid growth rate, its adaptability to a wide range of climate and soil conditions, its high water use efficiency, and its low fertilizer requirement (Geng et al., 1989; Amaducci et al., 2004; Prasad et al., 2007; Bennett and Anex, 2009; Wu et al., 2010). Dos Passos Bernardes et al. (2014) tested sweet sorghum in eastern Canada and reported DM yields of 15.8 Mg DM ha-1 and WSC concentration of 231 g kg DM-1. Yields from 17 to 25 Mg DM ha-1 were reported in Italy (Barbanti et al., 2006), while they reached 21.8 Mg DM ha-1 in Florida (Erickson et al., 2012) and 17.6 Mg ha-1 in Texas (Tamang et al., 2011). Barbanti et al. (2006) and Erickson et al. (2012) did not observe any significant differences in sweet sorghum DM yield when varying fertilizer N rates, while Tamang et al. (2011) observed a quadratic response only one year out of two. Experiments in Iowa and Colorado also suggested that N rates had only a limited influence on DM, ethanol, and WSC yields, which reached up to 6 Mg WSC ha-1 even without N fertilization (Smith and Buxton, 1993). Hence, based on results from the literature, high DM yields were expected for both species under the climatic conditions of eastern Canada, with a modest response to increasing N rate. Higher WSC and ethanol yields were expected for sweet sorghum than for sweet pearl millet.
Animal manure has been tested as a fertilizer with non-sweet hybrids of pearl millet and sorghum. Most studies, conducted in Africa, reported additive and even synergetic effects on yield from the combination of manure and low rates of mineral N fertilizer (Bayu et al., 2006; Amujoyegbe et al., 2007; Akponikpe et al., 2008). However, the information is limited on the effect of manure on sweet hybrids (Penn et al., 2014). Manure is abundant in eastern Canada, and liquid swine manure (LSM) has proved to be as efficient as mineral N fertilizer when applied to grain corn and spring wheat (Triticum aestivum L.) and immediately incorporated to minimize NH3 volatilization (Chantigny et al., 2008; Rieux et al., 2013). Woli et al. (2013) reported fertilizer N equivalence (FNE) to mineral fertilizer of 61 and 64% for LSM applied to corn, in Iowa. In contrast, liquid dairy manure (LDM) resulted in lower yields for spring wheat when compared to mineral N fertilizer in eastern Canada (Rieux et al., 2013). Jokela (1992) calculated FNE ranging from 27 to 44% for semi-solid dairy manure when applied to silage corn, in Vermont. For sweet pearl millet and sweet sorghum, we then expected
39 yields to be similar between LSM and mineral N fertilization, but to be reduced under LDM fertilization.
We are not aware of studies that compared splitting the N application to a single N application at seeding for sweet pearl millet and sweet sorghum. Previous studies on sweet pearl millet in eastern Canada used split N application at seeding and tillering (Bouchard et al., 2011; Leblanc et al., 2012; Crépeau et al., 2013; Dos Passos Bernardes et al., 2014). Most of the recent studies on sweet sorghum also used a split N application (Steduto et al., 1997; Barbanti et al., 2006; Zhao et al., 2009; Propheter et al., 2010; Han et al., 2011; Erikson et al., 2012; Dos Passos Bernardes et al., 2014). For grain corn in Quebec (Canada), a split N application is recommended (CRAAQ, 2010). This practice contributes to minimizing the risk of nitrate leaching (Gehl et al., 2005). Therefore, we hypothesized that a split N application in sweet pearl millet and sweet sorghum would result in greater yield than a single application at seeding.
The objectives of this study were (i) to compare sweet pearl millet and sweet sorghum for DM, WSC, and predicted ethanol yields; and (ii) to determine their response to increasing mineral N rate (0 to 160 kg N ha-1), to N source (mineral vs. organic), and to single vs. split applications of mineral fertilizer (80 kg N ha-1), at two sites with contrasted temperatures in eastern Canada.
40
Materials and methods
Sites and species descriptionThis study was carried out at two experimental sites with different temperature regimes. Ecozones of Canada are described in the National ecological framework for Canada (ESWG, 1995) and the climatic index used to compare temperatures among sites was calculated as per Brown and Bootsma (1993) and is expressed in corn heat units (CHU). The first site, located at Sainte-Anne-de-Bellevue (Québec, Canada, 45˚26’N, 73˚56’W), accumulates 2901 to 3100 CHU during the growing season (CRAAQ, 2012) and is part of the Mixedwood Plains ecozone (MWP). The second site, located at Saint-Augustin-de-Desmaures (Québec, Canada, 46˚44’N, 71˚31’W), accumulates 2501 to 2700 CHU during the growing season (CRAAQ, 2012) and is part of the Boreal Shield ecozone (BS). Soil types were a St. Bernard sandy loam (coarse-loamy, mixed, nonacid, calcareous, frigid Eutrochrept) at the MWP site, and a St. Antoine sandy loam (fine-loamy, mixed, acid, frigid Haplorthod) at the BS site. Selected soil characteristics are detailed in Table 1-1. At MWP, air temperature and rainfall (Fig. 1-1) were retrieved from the weather station of the Pierre Elliott Trudeau airport (45˚28’N, 73˚45'W), located at approximately 15 km from the experimental field. At BS, air temperature and rainfall were monitored onsite (Fig. 1-1).
At both sites, sweet pearl millet hybrid CSSPM7 (AERC Inc., Delhi, ON, Canada) and sweet sorghum hybrid CSSH45 (AERC Inc., Delhi, ON, Canada) were grown. These hybrids have been developed for the climatic conditions of eastern Canada (AERC, 2014). Both species were grown in 2010 and 2011 at BS, and in 2011 and 2012 at MWP. For the two consecutive years at each site, the species were grown on different plots in adjacent fields. The previous crops grown on these experimental plots were spring barley (Hordeum vulgare L.) for both years (2010 and 2011) at BS, and corn in 2011 and oats (Avena sativa L.) in 2012 at MWP.
Experimental set-up and crop management
A split-plot factorial design with four replications was set, with species as the main factor (main plots) and N fertilization treatments as the sub-factor (subplots). Subplots included seven 5-m rows at MWP (area of 6.3 m2), and nine 6-m rows (area of 9.7 m2) at BS. Row spacing was 0.18 m. Seedbed preparation consisted of mouldboard ploughing in the fall, harrowing in the spring to stimulate weed
41 germination, and a final harrowing to kill weeds prior to seeding. The seeding rate was 10 kg ha-1 of pure live seeds. Seeding was performed at a depth of 2.5 cm, as soon as soil temperature reached 12°C (AERC, 2014), using a Wintersteiger plot seeder (Wintersteiger, Salt Lake City, UT). Seeding and harvesting dates are shown in Fig.1-1.
Bentazone (3-isopropyl-1H-2.1.3-benzothiadiazin-4(3H)-one 2.2-dioxide) was applied at a rate of 1.08 kg active ingredient ha-1 between the 3- and 6-leaf stage for both species to suppress dicotyledon weeds. At BS, glyphosate (N-(phosphonomethyl-glycine) was applied in spring 2010 (0.89 kg active ingredient ha-1) to control weeds. Hand weeding of the sweet sorghum plots was done at the 10-leaf stage at both sites, while the dense crop cover repressed most of the weeds in sweet pearl millet plots, and no weeding was necessary.
Fertilization treatments
Nitrogen fertilization treatments included calcium ammonium nitrate (27-0-0) at rates of 0, 40, 80, 120 and 160 kg N ha-1, with 40 kg N ha-1 broadcast at seeding and the remaining N sidedressed at the 4- leaf stage for the treatments receiving 80, 120 and 160 kg N ha-1. The mineral N treatments are referred to hereafter as 0, 40, 80-S, 120-S, and 160-S, where S stands for “split”. Two organic N sources, LSM and LDM, were broadcasted at seeding, at a target rate of 80 kg ha-1 of total N, based on preliminary analysis of their total N content (treatments referred to as LSM80 and LDM80). Manures were sampled again during field application and analyzed again for total N. The actual amounts of N applied varied between 75.0 and 89.8 kg N ha-1 (Table 1-2). The last mineral N fertilization treatment was calcium ammonium nitrate broadcast at 80 kg N ha-1 at seeding only (treatment referred to as 80) for a comparison with organic N sources (80 vs. LSM80 and LDM80) and a comparison with the split application (80 vs. 80-S).
Phosphorus was applied as triple superphosphate (0-46-0) and potassium as potassium chloride (0-0- 60) based on soil analyses and local recommendations (CRAAQ, 2010). Therefore, 40 kg P2O5 ha-1 and 60 kg K2O ha-1 were annually applied at MWP, and 20 kg P2O5 ha-1 and 40 kg K2O ha-1 at BS. No P and K were added to the plots fertilized with organic N sources (LSM80 and LDM80) because the slurries already contained these nutrients. All fertilizers (mineral and organic) applied at seeding were broadcast on the soil surface and incorporated into the top 5 cm of soil with one pass of an harrow within two hours of application. Selected characteristics of manures are given in Table 1-2.
42
Determination of plant DM and WSC yields
Both species were harvested when 3/4 of the inflorescence had emerged on at least 75% of the plants at the MWP site, and on at least 50% of the plants at the BS site. Five rows of 3.5 m in length in the center of each plot were hand harvested to a 5-cm stubble height. At BS in 2010, only two rows of 2 m in length were harvested in the center of each plot because of lodging.
The harvested biomass was weighed, chopped, and two fresh subsamples of 250 g were taken from each plot. The first subsample was dried at 55 °C until constant weight to determine DM concentration. The WSC concentration was determined on the second subsample, which was first heated for one minute in a microwave oven to quickly reduce plant metabolic activity (Pelletier et al., 2010), dried at 55 °C until constant weight, and ground with a Wiley mill (Standard model 3, Arthur H. Thomas Co., Philadelphia, PA) to pass a 1-mm screen. Approximately 500 mg of this ground material was incubated in 12 mL of deionized H2O at 80°C for 20 min for WSC extraction. Tubes were then incubated overnight at 4°C and subsequently centrifuged 10 min at 1500 g. One mL of the supernatant was collected, and WSC (sucrose, glucose, and fructose) were analyzed using an ACQUITY Ultra Performance Liquid Chromatography (UPLC) system controlled by the Empower II chromatography data software (Waters, Milford, MA). Elution was performed at 35°C at a flow rate of 0.25 mL min−1 with the following gradient of eluent A (80% acetonitrile with 0.1% NH4OH) and eluent B (30% acetonitrile with 0.1% NH4OH): 70% A and 30% B from 0 to 15 min, 30% A and 70% B from 15 to 17.5 min, and 70% A and 30% B from 17.5 to 20 min. The WSC were separated on an ACQUITY UPLC BEH AMIDE 1.7µm (2.1 x 100mm) column preceded by a VanGuard (2.1 x 5mm) pre-column, and detected on an Electric Light Scattering Detector (ELSD). The drift tube was set at 50°C in the cooling mode. Samples were kept at 4°C in the sample manager throughout the analysis. Peak identity and quantity of sucrose, glucose, and fructose were determined by comparison to standards. Sucrose, fructose, and glucose concentrations were added to determine the total WSC concentration. WSC concentration was multiplied by the crop DM yield to obtain WSC yield.
Calculations and statistical analyses
The theoretical ethanol yield (equation 1) was calculated for sweet pearl millet and sweet sorghum as per Smith and Buxton (1993) and Schmer et al. (2012), assuming 100% WSC extraction efficiency from the stalks by pressing and 100% WSC conversion to ethanol:
43 (1) Theoretical ethanol yield (L ha-1) = ((Glucose + fructose yield (Mg ha-1) × 510 kg ethanol Mg-1)
+ (Sucrose yield (Mg ha-1) × 537 kg ethanol Mg-1 )) / 0.789 kg ethanol L-1 ethanol
According to many authors (Bryan et al., 1985; Weitzel et al., 1989; Mask and Morris, 1991; Putnam et al., 1991; Propheter et al., 2010), only 50% to 60% of the WSC are expected to be extracted from the stalks by pressing, and 85% to 95% of those will be converted to ethanol at the biorefinery (Bryan et al., 1985; Prasad et al., 2006; Zhao et al., 2009; Wu et al., 2010; Yu et al., 2012). Hence, a predicted ethanol yield (equation 2) was calculated considering a conservative WSC extraction efficiency of 50% from pressing of the biomass, and a 90% WSC conversion rate to ethanol:
(2) Predicted ethanol yield (L ha-1) = Theoretical ethanol yield (L ha-1) × 50% WSC extraction efficiency × 90% WSC conversion rate
Data were analyzed separately for each site. Replications and years were considered random effects, whereas species and N treatments were considered fixed effects. Analyses of variance were performed using the MIXED procedure in SAS (SAS Institute, 2003) for the dependent variables: DM yield, WSC concentration, WSC yield, and theoretical and predicted ethanol yields. Data normality was verified using the UNIVARIATE procedure, and the Shapiro-Wilk test (Shapiro and Wilk, 1965) was used to determine whether the residuals were normally distributed. The homogeneity of variance was verified visually with graphics of residuals. Standard error of the means (SEM) is reported. Quantitative, single-degree of freedom (df) contrasts were used to test the response of DM, WSC, and ethanol yields as a function of mineral N rate. Single-df contrasts were also used to test the effect of N source (80 vs. LSM80 and LDM80), to compare the two organic N sources (LSM80 vs. LDM80), and to compare the effect of split vs. single application of mineral N (80 vs. 80-S). Statistical significance was postulated at P ≤ 0.05.
After being tested with quantitative, single-df contrasts, the responses of DM and WSC yields to mineral N rate (0, 40, 80-S, 120-S, and 160-S) were described with a quadratic response curve for each site, as in equation 3:
(3) Y = a + bN + cN2
where Y is the DM or WSC yield (Mg ha-1), N is the amount of fertilizer applied (kg N ha-1), and a, b, and c are estimated parameters. Linear regressions were used to establish relationships among
44
variables with the FIT directive of the GENSTAT 14 statistical software package (VSN International, 2011). A linear parallel curve analysis with grouped data was performed, as in Ziadi et al. (2008), to determine if the response to increasing mineral N rate differed between species. The curve analysis was performed with a step-wise addition of factors to the model (N rate, then species, then species N rate interaction): (i) one equation across species was initially calculated to describe the average response to N rate, (ii) separate intercept parameters were estimated for each species to determine the distance between parallel response curves and, (iii) separate slope parameters were estimated for each species (species N rate interaction). Each time a new factor was added to the model, the change in mean squares was tested for statistical significance (P ≤ 0.05).
The N rate required to reach maximum DM yield (Nmax DM) or maximum WSC yield (Nmax WSC) was estimated using the fitted parameters of the quadratic model. Because sweet pearl millet and sweet sorghum are new species in eastern Canada and do not have a market value, the economically optimal N rate could not be calculated. The Nmax rate (equation 4) was therefore calculated as in Tamang et al. (2011):
(4) Nmax = - b / 2c
The fertilizer N equivalence (FNE) of LSM and LDM were determined by entering DM yield for each manure in equation 3. This gave an equivalent mineral fertilizer N rate for each manure (Nfert), which was divided by the total N rate applied with the manure (Ntot manure) (average of the two years at each site, Table 1-2), as per equation 5:
45
Results and discussion
Dry matter yieldThe average DM yield of both species was 16.5 Mg DM ha-1 at the MWP site, and 11.3 Mg DM ha-1 at the BS site(Table 1-3). Similar differences among sites were also reported by Bouchard et al. (2011) and Leblanc et al. (2012) for DM yield in sweet pearl millet. Higher daily temperature at MWP likely stimulated the growth and development of those two C4 species (Mask and Morris, 1991). Mean daily temperatures from seeding to harvest were 21.5°C in 2011 and 21.9°C in 2012 at MWP, and 19.3°C in 2010 and 18.6°C in 2011 at BS (Fig. 1-1). Therefore, the accumulation of CHU from seeding to harvest was slightly higher at MWP, with 2189 CHU in 2011 and 2225 CHU in 2012, whereas 2095 CHU were accumulated in 2010 and 1940 in 2011 at BS (Fig. 1-1). To prevent lodging at BS, the two species were harvested before the desired maturity level, with only 50% of the plants having 3/4 of the inflorescence emerged. This might explain the lower yield recorded at BS, as Dos Passos Bernardes et al. (2014) found that delaying harvest beyond that stage resulted in greater DM yield in sweet pearl millet and sweet sorghum.
Differences in DM yield were not significant between species at MWP but were significantly higher with sweet pearl millet than sweet sorghum at BS (Tables 1-3 and 1-4). Sweet pearl millet DM yields, averaged across N treatments, were 11.6 Mg ha-1 at BS and 17.1 Mg ha-1 at MWP (Table 1-3). This is in agreement with other studies conducted in eastern Canada, where sweet pearl millet yielded