Variation in soil carbon under contrasting biodiesel feedstock crops

Pedobiologia 56 (2013) 61–67

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Pedobiologia - International Journal of Soil Biology journal homepage: www.elsevier.de/pedobi

Variation in soil carbon under contrasting biodiesel feedstock crops Roger T. Koide ∗ , Matthew S. Peoples, Emma T. Matheson Department of Horticulture and Graduate Program in Ecology, The Pennsylvania State University, University Park, PA 16802, USA

a r t i c l e

i n f o

Article history: Received 24 August 2012 Received in revised form 15 October 2012 Accepted 13 November 2012 Keywords: Biodiesel Canola Carbon savings Soil carbon Soil organic matter Soy

a b s t r a c t There is considerable interest in both soy and canola as biodiesel feedstock crops because of the net reduction in CO2 emissions resulting from the use of biodiesel in place of petroleum diesel. Whether these two crops differ in net CO2 savings is unknown, in part because of our ignorance of their impact on soil C. We, therefore, monitored soil C for three years in an experiment that included both soy and canola rotations. We found that soil C concentrations were significantly lower under canola than soy, and that the difference represented as much as 64% of the C savings of soy biodiesel over petroleum diesel. We tested two hypotheses that could explain this difference in soil C. First, because canola can acidify the soil, we determined whether a reduction in inorganic C (as carbonate) in canola plots could account for the soil C difference. Carbonate concentration did not differ significantly in soy and canola plots. Second, we determined whether soil organic matter concentration could account for the soil C difference. Soil organic matter concentration was significantly lower in canola than in soy plots, accounting for the difference in soil C. We further hypothesized that because soy is mycorrhizal and canola is not, soy soils should contain higher concentrations of glomalin, a recalcitrant substance produced by mycorrhizal fungi, and that this could help to explain the difference in soil organic matter. Glomalin concentrations were significantly lower in canola plots, but this difference accounted for only a fraction of the total soil C difference. Our results suggest that a proper accounting of life cycle C savings of biodiesel when used in place of petroleum diesel must consider soil C. © 2012 Elsevier GmbH. All rights reserved.

Introduction The use of biodiesel in place of petroleum diesel has the potential to significantly reduce net CO2 emissions, primarily because the CO2 produced during biodiesel combustion is recycled via photosynthesis by the feedstock crop (Sheehan et al. 1998). However, even relatively small changes in soil C concentration may have dramatic impacts on the overall C balance of the biodiesel because of the vast size of the soil C pool. The extent to which net CO2 emissions are reduced by using biodiesel in place of petroleum diesel may, therefore, depend strongly on feedstock crop effects on soil C. While we now appreciate the need to account for changes in soil C as a consequence of land use conversion to biofuel production (Zenone et al. 2011), the removal of crop residues (Kochsiek and Knops 2012) or the choice between perennial and annual biofuel systems (Gelfand et al. 2011), no comparisons have been made of the effects of alternative annual biodiesel feedstock crops on soil C. In temperate climates both soy and canola are commonly grown as oilseed crops. In the United States, more soy (Glycine max) is produced than any other oilseed crop (USDA 2011a). Consequently, soy

∗ Corresponding author at: Department of Biology, Brigham Young University, Provo, UT 84602, USA. Tel.: +1 801 422 6650. E-mail address: [email protected] (R.T. Koide). 0031-4056/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.pedobi.2012.11.002

oil is currently the most commonly used feedstock for biodiesel production in the United States (USDOE 2011). In Western Europe, canola (rape, Brassica napus L.) is the most common biodiesel feedstock crop (Haas 2005). While canola is not yet grown to a great extent in the United States (USDA 2011a), it is an attractive crop because nearly three times as much oil per hectare can potentially be derived from canola compared to soy (Gui et al. 2008; Pahl 2008). Moreover, there is currently a higher market price for canola oil compared to soy oil (USDA 2011b). The net reduction in CO2 emissions from the use of biodiesel in place of petroleum diesel depends on several factors that vary with feedstock crop. For example, while nitrogen-fixing legumes, such as soy, may require little or no nitrogen fertilizer, the requirement for nitrogen fertilization of canola adds to net CO2 emissions because of the energy used to produce, ship and apply the fertilizer (Haas et al. 2001; Fore et al. 2011). Moreover, soy and canola may also differ significantly in their capacity to contribute to new soil C or in their effects on existing soil C. Soy, like most terrestrial plant species, establishes a symbiosis with mycorrhizal fungi (Jones 1924; Ross and Harper 1970) while canola, as with most members of the Brassicaceae, does not (Hirrel et al. 1978; Mozafar et al. 2000), due, in part, to the production of anti-fungal mustard oils (Schreiner and Koide 1993). A large fraction of the C captured by a host plant in the process of photosynthesis enters the soil via its mycorrhizal fungi (Treseder and Allen 2000), some of which occurs in the form

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of a glycoprotein, glomalin that may contribute significantly to soil organic C (Rillig et al. 2001; Rillig 2004). While the fungi do not produce large quantities of glomalin (Driver et al. 2005), glomalin may still contribute significantly to soil C because of its relative recalcitrance to decomposition (Rillig 2004). Moreover, both glomalin and mycorrhizal fungal hyphae are known to improve soil aggregation (Miller and Jastrow 1990; Rillig 2004), and soil aggregation may improve soil C sequestration by protecting organic matter from rapid decomposition (Tisdall and Oades 1979; Elliott and Coleman 1988; Jastrow 1996; Jastrow and Miller 1997; Tisdall et al. 1997; Jastrow et al. 2005). A direct comparison of the C concentration of soils under soy and canola crops has not yet been made. This comparison is necessary to determine unequivocally whether these two annual oilseed crop species differ in their ability to reduce net CO2 emissions compared to petroleum diesel. We, therefore, compared the properties of soils from under canola and soy crops, and continued to follow the soils for two more years of maize production because canola is typically grown in a three- or four-year rotation to suppress disease (George et al. 2008; Kandel and Knodel 2011). Planting a mycorrhizal winter cover crop, a common practice in some regions such as the Chesapeake Bay watershed (Hively et al. 2009), can ameliorate the negative impact of winter fallow on mycorrhizal fungal populations (Boswell et al. 1998; Kabir and Koide 2000; Kabir and Koide 2002). Because mycorrhizal fungi contribute to soil organic C via their production of glomalin, we also determined the effects of winter cover cropping on the same soil properties.

Materials and methods Site information and field operations This study was conducted from 2008 to 2010 within a 0.53 ha field located at the Russell Larson Research and Education Center at Rock Springs, PA, USA, 40◦ 42 45.95 N, 77◦ 57 28.83 W. The soil is Hagerstown series (fine, mixed, semiactive, mesic Typic Hapludalfs, USDA-NRCS 2011) with a silt loam surface texture and subsurface textures of silty clay loam and silty clay. In the four years preceding this experiment the entire field had been uniformly planted to the following crops: 2004 wheat (Triticum aestivum L.), 2005 fallow with wheat planted in October 2005 as a winter cover crop, 2006 squash (Cucurbita pepo L.), 2007 wheat. During those years, all field operations, including cultivation and fertilization, were applied uniformly across the entire field. Spring 2008. In spring 2008, soil was composited from several, randomly drawn subsamples taken to 15 cm in the field in which this research was performed. Standard fertility testing of the soil was performed by the Pennsylvania State University Agricultural Analytical Service Laboratory. Soil pH was determined in a 0.01 M CaCl2 aqueous solution and available soil P, K, Ca and Mg were determined by inductively coupled plasma spectroscopy on Mehlich III extractions (Wolf and Beegle 1995). Cation exchange capacity was determined by summation (Ross 1995).

On 18 April 2008 we cultivated the entire field with an S-tine cultivator to prepare the soil for planting canola and soybean, but in subsequent years the maize was planted without tillage. On 19 April 2008 the field was divided into 8 blocks (n = 8 replicates), each consisting of 4 plots (6 × 18 m) for each of the four rotations (Table 1), which were randomly arranged within each block (randomized complete block experimental design). For both the canola/winter-fallow and canola/wintercover rotations we applied sulfur-coated urea (39-0-0-12) with a walk-behind spinner-spreader at approximately 2.9 kg plot−1 , supplying approximately 112 kg N ha−1 prior to planting. Canola, Brassica napus L. (DeKalb 34-65, Roundup Ready, open-pollinated) was drilled on 18 cm row spacing in plots of the canola/winterfallow and canola/winter-cover rotations on 24 April 2008 at the rate of 6.7 kg ha−1 (315,000 seeds kg−1 ). Plots of the soy/winterfallow and soy/winter-cover rotations received no fertilizer but were planted to soybean, Glycine max (L.) Merr, (Asgrow AG 3006 Roundup Ready) at the rate of 56 kg ha−1 (7900 seeds kg−1 ) on 7 May 2008. Weed suppression throughout the field site was accomplished as needed uniformly across the entire field site (irrespective of rotation) with glyphosate herbicide at the rate of 5 L ha−1 active ingredient. Summer/Autumn 2008. Canola was harvested from canola/winter-fallow and canola/winter-cover rotations on 4 August 2008. Soy was harvested from the soy/winter-fallow and soy/winter-cover rotations on 15 October 2008. Wheat (Triticum aestivum L.) was planted into plots of the soy/winter-cover rotation (soy in the first summer) and the canola/winter-cover rotation (canola in the first summer) on 23 October 2008 at the rate of 110 kg ha−1 . No fertilizer was applied to the wheat. Plots of the soy/winter-fallow and the canola/winter-fallow rotations were winter-fallowed after harvest of soy and canola. Spring 2009. On 4 May 2009, the entire site was treated with Ignite herbicide (Bayer) to kill the wheat and the canola that had successfully overwintered. On 12 May 2009, maize (DeKalb DKC 57-79 RR2/YGPL) was planted into each plot of all rotations in rows 76 cm apart. On 22 May 2009 urea fertilizer was applied at the rate of 206 kg N ha−1 . Autumn/Winter 2009. The maize was harvested on 30 November 2009. On 1 December 2009 we planted cereal rye (Secale cereale) in the plots of the soy/winter-cover and canola/winter-cover rotations at approximately 64 kg ha−1 . Rye is generally considered to be superior to wheat at germinating and growing at cold temperatures (Clark 2007), so it was used in place of wheat because of the late harvest of the maize. No fertilizer was applied to the rye. Plots of soy/winter-fallow and canola/winterfallow rotations were winter-fallowed after the maize harvest. Spring/Summer/Autumn 2010. On 4 May 2010 we treated all plots with glyphosate to kill the rye. On 7 May we again planted maize (DeKalb DKC 53-17 VT3, 103 d maturity) in the plots of all rotations with 76 cm row spacing. A starter dose of urea-ammonium nitrate (UAN 32-0-0) was applied at the rate of 39 kg N ha−1 at planting time. On 29 June 2010 we side-dressed with an additional 112 kg N ha−1 as UAN 32-0-0. The maize was harvested on 26 October 2010.

Table 1 The four crop rotations. We utilized a randomized complete block design, with eight blocks. Rotation

Designation

1 2 3 4

Soy/winter fallow Canola/winter fallow Soy/winter cover Canola/winter cover

Sequence of crops in rotation Summer 2008

Winter cover 2008–2009

Summer 2009

Winter cover 2009–2010

Summer 2010

Soy Canola Soy Canola

Fallow Fallow Wheat Wheat

Maize Maize Maize Maize

Fallow Fallow Rye Rye

Maize Maize Maize Maize

R.T. Koide et al. / Pedobiologia 56 (2013) 61–67

Standing root biomass, root production and root turnover Three soil cores (2 cm diameter) were taken from random locations within each plot to approximately 12 cm from each plot of soy/winter-cover and canola/winter-cover rotations on 27 May 2008, 30 June 2008, 26 July 2008, 27 August 2008, 30 September 2008 and from each plot of all rotations on 31 March 2009, 16 June 2009, 2 June 2010. All cores from a single plot were pooled to constitute a single sample. Standing root biomass was determined by placing three field-moist soil cores in 5% sodium hexametaphosphate overnight to loosen adhering soil, then wet-sieved to rinse and recover the roots, which were dried to constant weight at 70 ◦ C. To assess root production, we made and installed PVC-coated, fiberglass mesh (1 mm mesh) bags with into the plots of rotations 3 (soy) and 4 (canola). The bags had dimensions of 3.0 cm diameter and 12.0 cm length, with an open top. We used a soil corer of the same diameter at three random locations within the plots to remove soil and create a 12 cm deep hole into which we installed the bags. The bags were then filled with the approximately 85 ml of soil originally removed with the corer. The soil in the bag was packed so that it was level with the soil in the plots. For purposes of identification, an aluminum tag was tied to the bag with a copper wire. Bags were first installed on 12 June 2008. The bags were collected and new bags inserted into the same holes on 30 June 2008, 28 July 2008, 27 August 2008, 30 September 2008. Bags were no longer installed in plots of the canola/winter-cover or canola/winter-fallow rotations at the last three dates because the canola was harvested on 4 August 2008. Upon collection each bag was placed in 5% sodium hexametaphosphate overnight, then wetsieving was used to recover the roots, which were dried to constant weight at 70 ◦ C. Root turnover was calculated according to Gill and Jackson (2000). Statistical analyses Data were subjected to analysis of variance for a randomized complete block design with eight blocks (replicates). Response variables included total soil C concentration, soil loss on ignition, total glomalin, standing root biomass, root production, and water-stable

Results In 2008, the mean soil pH for the entire field was 6.2. Mean soil P, K, Mg and Ca availabilities were 40, 105, 133 and 2095 ppm, respectively. Cation exchange capacity was 11.9 mequiv per 100 g. During the 2008 growing season (the year soy and canola were grown), soil total C concentration was significantly (p = 0.0044) greater in soy plots than in canola plots, date was not a significant factor and the interaction between date and crop was not significant (Fig. 1a). For the three sampling dates in 2009 and 2010, the plots that were formerly planted to soy continued to have significantly (p = 0.007) greater soil total C concentrations than those formerly planted to canola, soil total C significantly (p = 0.010) declined with time, and cover cropping did not have a significant effect. There were no significant interactions among factors (Fig. 1b). Soils collected on 30 June 2008 and 28 July 2008 from soy and canola plots had mean carbonate C concentrations of 0.749 mg g−1 soil. Neither crop (canola vs. soy) nor date was significant factor with respect to carbonate C, and the interaction between crop and date was not significant.

Canola Soy

(a)

15

Soil C (mg g-1)

Six soil cores (2 cm diameter) were taken from random locations within each plot to approximately 12 cm from each plot of soy/winter-cover and canola/winter-cover rotations on 27 May 2008, 30 June 2008, 26 July 2008 and 30 September 2008 and from each plot of all rotations on 31 March 2009, 16 June 2009, 2 June 2010. The six cores from each plot were pooled to constitute a single sample, passed through a 2.0 mm sieve and allowed to dry in paper bags. Soil total C concentrations for all soil collections were determined by the University of Connecticut Soil Nutrient Analysis Lab (Storrs, CT, USA) using an Elementar Vario-Max instrument. Carbonate concentrations for soils sampled on 30 June 2008 and 28 July 2008 were determined using the acetic acid method of Loeppert et al. (1984). Soil organic matter concentration was estimated on a subsample of soil from each plot for 27 May, 26 July and 30 September in 2008, and 31 March 2009, 16 June 2009, 2 June 2010 as the proportion of soil dry weight lost on ignition at 550 ◦ C for 4 h (Heiri et al. 2001). Total glomalin was characterized for all soil collections from citrate extracts of soil as Bradford reactive soil protein according to Rosier et al. (2006). We detected the protein colorimetrically using the ratiometric method of Zor and Selinger (1996). A subsample of <2.0 mm soil was passed through a 1 mm sieve to isolate the 1–2 mm fraction, which was used for analysis of water-stable aggregates according to Angers and Mehuys (1993).

aggregates. For data collected in 2008 the independent variable was ‘crop in 2008’ (canola vs. soy). For data collected in 2009 and 2010 the independent variables were ‘crop in 2008’ (canola vs. soy) and ‘winter cover cropping’ (present vs. absent). When measurements were made over time, the data were analyzed with a repeated measures analysis of variance. Correlations were determined using the correlation matrix module of Statistica version 6 (Statsoft, Inc., Tulsa, OK, USA). In all cases, effects or correlations were considered significant only when p ≤ 0.05.

10

5

0 27-May-08 30-Jun-08 28-Jul-08 30-Sep-08

20

Soil C (mg g-1)

Soil total C, carbonate C, organic matter, glomalin, aggregate stability

63

Canola, fallow Canola, cover crop Soy, fallow Soy, cover crop

(b)

15 10 5 0 31 March 2009

16 June 2009

2 June 2010

Date Fig. 1. Soil total C concentrations in canola and soy plots at four sampling dates in 2008 (a) and in former canola and soy plots at three dates in 2009–2010 (b). n = 8. Error bars represent ± 1 s.e.m.

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50

22

(a)

Canola Soy

20

Soil C (mg g-1)

Soil loss on ignition (mg g-1)

60

40 30 20

27-May-08

28-Jul-08

Canola, fallow Canola, cover crop Soy, fallow Soy, cover crop

30-Sep-08

(b)

40

45

50

55

60

Loss on igntion (mg g-1) Fig. 3. Relationship between soil total C and soil organic matter measured as loss on ignition. y = 0.372x − 0.14. p < 0.0001, r = 0.75. Dark circles = soy plots, light circles = canola plots.

40

20 10 0 31 March 2009 16 June 2009

2 June 2010

Date Fig. 2. Soil organic matter as assessed by loss on ignition in canola and soy plots at three sampling dates in 2008 (a) and in former canola and soy plots at three dates in 2009–2010 (b). n = 8. Error bars represent ± 1 s.e.m.

In 2008 soil loss on ignition was significantly (p = 0.008) greater in soy plots than in canola plots, date was not a significant factor and the interaction between date and crop was not significant (Fig. 2a). For the three sampling dates in 2009 and 2010 (Fig. 2b), the plots that were formerly planted to soy continued to have significantly (p = 0.006) greater soil loss on ignition than those formerly planted to canola, and soil loss on ignition significantly (p = 0.032) declined with time. There was a significant interaction between cover cropping and date with respect to loss on ignition (Fig. 2b), with a positive effect of cover cropping on 31 March 2009, but negative effects of cover cropping on the two subsequent dates (16 June 2009 and 2 June 2010). Soil total C and soil loss on ignition were significantly correlated (Fig. 3). The Pearson correlation coefficient, r, for this relationship was 0.75. In 2008 total glomalin (Fig. 4a) was significantly (p = 0.019) higher in soy plots than in canola plots, total glomalin exhibited a significant (p = 0.003) increase as the season progressed, but there was no significant interaction between sampling date and crop. In 2009 and 2010, total glomalin was not significantly affected by date or by cover cropping, but it was nearly significantly (p = 0.057) greater in former soy plots than in former canola plots (Fig. 4b). During 2008 there was a significant interaction (p < 0.0001) between crop (canola vs. soy) and date for standing root biomass (Fig. 5a). Standing root biomass was much greater for soy than for canola on all dates except 27 May. There was a significant interaction (p < 0.0001) between crop (canola vs. soy) and date for root production during the 2008 growing season (Fig. 5b). Root production was significantly higher for soy than canola for all periods except for the period ending 30 June, when the opposite was true. When canola and soy were analyzed separately, root production was significantly affected by date for both soy and canola,

but the temporal pattern was different for the two crops; maximum root production occurred approximately 1 month later in soy plots than in canola plots. Cumulative root production during the period 27 May to 30 September was similar for the two crops: 74.1 ␮g d−1 cm−3 for canola and 70.7 for soy. Root turnover (d−1 ) was not significantly (p = 0.18) affected by crop. The root turnover means and standard errors were 0.131 (0.055) and 0.237 (0.053) for canola and soy, respectively. In 2008 there was a significant interaction between crop (canola vs. soy) and date for the fraction of soil comprising 1–2 mm waterstable aggregates. At the first two dates, canola soils had lower

Canola Soy

Total glomalin (mg g-1)

30

(a)

2.5 2.0 1.5 1.0 0.5 0.0

27-May-08 30-Jun-08 28-Jul-08 30-Sep-08 3.0

Total glomalin (mg g-1)

Soil loss on ignition (mg g-1)

14

10 35

0

50

16

12

10

60

18

2.5

Canola, fallow Canola, cover crop Soy, fallow Soy, cover crop

(b)

2.0 1.5 1.0 0.5 0.0 2 June 2010

16 June 2009 Date

Fig. 4. Total glomalin for soil samples taken on four dates in 2008 (a) and three dates during 2009–2010 (b). n = 8. Error bars represent ± 1 s.e.m.

1200

(a)

Canola Soy

1000

Root production, 0-12 cm (µg cm-3 d -1)

Standing root biomass 0-12 cm µ( g cm-3)

R.T. Koide et al. / Pedobiologia 56 (2013) 61–67

800 600 400 200 0

80

(b)

60

40

20

0

27-May-08 30-Jun-08

28-Jul-08

27-Aug-08 30-Sep-08

Date

Water-stable soil aggreates, 1-2 mm (%)

Fig. 5. Standing root biomass (a) and root production (b) for canola and soy during the 2008 growing season. n = 8. Error bars represent ± 1 s.e.m.

30 Canola Soy

20

10

0

27-May-08

28-Jul-08

30-Sep-08

Date Fig. 6. Water-stable soil aggregates as a percentage of the 1–2 mm soil fraction for three dates in 2008. n = 8. Error bars represent ± 1 s.e.m.

levels of soil aggregation than soy soils, but at the last date the reverse was seen (Fig. 6). Discussion In many parts of the world, including much of the United States of America, both soy and canola can be grown as biodiesel feedstock crops. It is generally assumed that use of any biodiesel in place of petroleum diesel will significantly reduce net CO2 emissions because of the recycling of CO2 via photosynthesis of the feedstock crop. However, significant differences among biodiesel feedstock crops in net CO2 savings are possible. Life cycle analyses

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indicate that the need for N fertilizer by canola reduces the potential net energy yield when compared to soy, but this can be largely offset by canola’s potentially greater per acre oil yield (Smith et al. 2007; Fore et al. 2011). Energy use is obviously closely tied to carbon cycling when the source of energy is a fossil fuel. Thus, the net CO2 savings for canola-based biodiesel and soy-based biodiesel, when compared to petroleum diesel, are assumed to be approximately the same assuming the two crops do not deviate from each other in other ways that impact net carbon balance. We show here, however, that canola and soy may differ markedly in their effects on the soil C pool. In 2008 (the year in which soy and canola were grown), the top 0.15 m of soil in the soy plots contained an average of 0.111 mg more C g−1 soil than the soil in the canola plots. This difference in soil C between soy and canola plots must have been caused by inherent differences between soy and canola. For the four years prior to the current study, the field in which the study occurred had been used to grow crops that were planted and managed uniformly across the entire field. Therefore, it seems very unlikely that the differences in soil C between soy and canola plots observed in this study were caused by patchiness of the field due to its previous management that corresponded to the random distribution of the canola and soy plots in the current experiment. The 0.111 mg g−1 more C in soy plots than in canola plots is equivalent to a difference of 0.133 mg C cm−3 soil, assuming a soil density of 1.2 g cm−3 . If we assume that there were no significant differences in soil C concentration below 0.15 m depth, then a hectare of soy soil is calculated to contain 1.98 × 105 additional grams of C than canola soils. This difference is highly consequential. According to the diesel life cycle analysis of Sheehan et al. (1998), every 1.00 kg soy used to produce biodiesel saves 0.109 kg C (as CO2 emissions) when compared to the use of an equivalent energy value of petroleum diesel. The U.S. average soy yield of 2830 kg ha−1 for 2007–2011 (USDA-NASS 2012) would, therefore, save 3.09 × 105 g C ha−1 compared to using petroleum diesel. Because the net energy yield per hectare from canola is approximately that of soy (the energy required to fertilize canola offsets the potential for greater oil yields, see Fore et al. 2011), the reduction in soil C by canola relative to soy (1.98 × 105 g C ha−1 ) represents approximately 64% of the C savings of soy biodiesel over petroleum diesel. In other words, by planting canola instead of soybean, the majority of the CO2 saved by using soy biodiesel over petroleum diesel would potentially be lost due to the significant reduction of soil C. Obviously differences in soil C concentrations below 0.15 m would also influence the effect of crop on net carbon savings. In order to avoid diseases, canola is grown in three or four year rotations (George et al. 2008; Kandel and Knodel 2011). Even in the two years subsequent to soy and canola in which maize was grown, the two-year average soil C concentrations remained significantly lower in former canola soils than in former soy soils. The difference was smaller (0.0639 as opposed to 0.111 mg C g−1 ) than in the year soy and canola were grown (2008), but a difference of 0.0639 mg C g−1 still represents a reduction of soil C in former canola soils equivalent to 37% of the C savings of soy biodiesel over petroleum diesel. These results are consistent with those from other studies that demonstrate the need to account for variation in soil C associated with land use change to accommodate biofuel production (Zenone et al. 2011), the removal of crop residues as biofuel feedstock (Kochsiek and Knops 2012), or the choice between perennial and annual biofuel systems (Gelfand et al. 2011). Unless soil C is accounted for, we may never know if it is possible to achieve the hoped for CO2 savings associated with biofuels. We tested two hypotheses that explain how canola may have reduced soil C concentration. First, a reduction in soil inorganic C (as carbonate) in canola plots could contribute to the reduction in soil

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total C. When canola is phosphorus deficient, it releases protons, acidifying the rhizosphere by more than 2 pH units in some cases (Grinsted et al. 1982; Hedley et al. 1982). Because a reduction of soil pH could theoretically release carbonate C as CO2 , we assayed for carbonate in soils collected on 30 June and 28 July 2008. The mean carbonate C concentration was 0.74 mg C g−1 soil (approximately 5% of total soil C), but there was no significant effect of crop (soy vs. canola) on soil carbonate concentration. Thus, variation in carbonate C concentration between soy and canola soils cannot account for the measured difference in total soil C. Second, a reduced concentration of organic soil C in canola plots could contribute to the reduction in soil total C. Soil organic matter as a whole comprises approximately 45.8% C (Ball 1964). The difference in 2008 between canola and soy plots in soil organic matter was 2.72 mg g−1 soil (Fig. 3). Therefore, the calculated difference in C accounted for by soil organic matter alone is 1.25 mg C g−1 , quite similar to the difference in total soil C as measured by the Vario-Max instrument of 1.11 mg C g−1 . Using the same assumptions for the period 2009–2010, the calculated difference in C accounted for by soil organic matter alone is 0.64 mg C g−1 , while the actual difference in total soil C (by Vario-Max instrument) was also 0.64 mg C g−1 . Thus, differences in soil organic matter concentration between canola and soy plots (based on loss-on-ignition) appear to entirely account for the difference in total soil C. Differences between canola and soy plots in soil organic matter concentration could be caused either by variation in organic matter inputs or variation in decomposition rates. One of the organic matter inputs in most agricultural systems is that produced by mycorrhizal fungi. However, in this respect soy and canola are quite distinct. Soy is arbuscular mycorrhizal (Jones 1924; Ross and Harper 1970) while canola, as with most members of the Brassicaceae, is not (Hirrel et al. 1978; Mozafar et al. 2000). The hyphae of mycorrhizal fungi are not likely to contribute significantly to the soil organic matter pool because they turn over rapidly, with a half-life of a few to several days (Friese and Allen 1991; Rillig et al. 2001; Staddon et al. 2003). This inability of mycorrhizal hyphae to contribute to the sequestration of soil C or organic matter beyond a single season appears to be inconsistent with the persistent difference in soil C between former soy and former canola plots during the two years in which maize was grown. Arbuscular mycorrhizal fungi, however, also produce glomalin, a glycoprotein that may have a relatively long turn-over duration, allowing it to accumulate to relatively high concentrations in the soil (Rillig et al. 2001). There was a significant effect of crop on total glomalin concentration in 2008 and a nearly significant effect in subsequent years. Nevertheless, the differences in glomalin concentration between canola and soy plots were not large enough to account for more than a small fraction of the difference in total soil C concentration. Assuming total glomalin contains 45% C by weight (Schindler et al. 2007), averaged across 2008, 2009 and 2010, total glomalin accounted for only 6.7% of total soil C. Because the difference in soil C and organic matter was evident early in the growing season for soy and canola, we examined root production as it is expected to have a more immediate effect on soil C than shoot production. Root production occurred earlier for canola than for soy, and the total seasonal root production was actually slightly higher for canola than for soy. Calculated root turnover was not significantly different between the two crops. Thus differences in root productivity do not readily explain the differences in soil C or soil organic matter between soy and canola plots. We hypothesize, therefore, that the significant difference between canola and soy plots, which was observed as early as 27 May 2008 and consistently throughout the study, was caused by variation in decomposition of soil organic matter. We propose three possible causes of enhanced decomposition in canola plots relative to soy plots. First, enhanced decomposition of soil organic matter in canola plots relative to soy plots could have

been caused by a reduced level of soil aggregation (Tisdall and Oades 1979; Elliott and Coleman 1988; Jastrow 1996; Jastrow and Miller 1997; Tisdall et al. 1997; Jastrow et al. 2005) in canola plots in May and July of 2008. By September, however, the trend had reversed. The cause of the variation in soil aggregation between canola and soy crops remains unknown. Glomalin concentration was not positively correlated with aggregation (data not shown) as is sometimes observed (Wright et al. 1999; Wright and Anderson 2000). Second, priming is the phenomenon in which addition of fresh organic matter stimulates decomposition of organic matter already present (Kuzyakov 2010). Plant species differ in the capacity to prime organic matter decomposition (Fu and Cheng 2002), presumably because of differences in root chemistry, but also potentially due to differences in the timing of organic matter additions. Thus, enhanced organic matter decomposition in canola plots relative to soy plots could have been caused by the canola vegetation priming decomposition more than soy, either because of differences in chemistry or the timing of organic matter inputs. Canola was planted on 24 April 2008, 14 d prior to planting soy, and its peak root growth occurred approximately one month prior to that of soy. Third, decomposition may also have been stimulated to a higher degree in canola plots than in soy plots because canola was fertilized with urea prior to sowing. In some cases increased N fertility has increased soil organic matter decomposition (Jenkinson et al. 1985; Hobbie 2000; Vestgarden 2001) although the stimulatory effect of N fertilization is certainly not universal (Hyvönen et al. 2007). Clearly more research needs to be performed to test these new hypotheses regarding the causes of variation in organic matter concentration in canola vs. soy soils, and to determine the circumstances under which soil total C differs under canola and soy. Nevertheless, our results do demonstrate that reductions in soil C concentration by canola compared to soy may strongly influence the C balance of the resultant biodiesel. Our results further demonstrate that soil C concentration must be determined in order to properly evaluate the net consequences of biofuels for potential CO2 savings. Acknowledgements The authors thank Mark Antle, Robert Oberheim and Scott Harkcom for assistance with soil preparation, planting and harvesting, and Chris Fernandez, Glenna Malcolm, William Murray and Elizabeth Reid for soils collection and analysis and plant analysis, and the Pennsylvania Soybean Promotion Board, the North East SunGrant and the Department of Horticulture, Pennsylvania State University for financial support. Sponsors had no role in the writing of this report, in the design of the experiment, or in the collection, analysis or interpretation of data. References Angers, D.A., Mehuys, G.R., 1993. Aggregate stability to water. In: Carter, M.R. (Ed.), Manual on Soil Sampling and Methods of Analysis. CRC Press, Boca Raton, FL, USA, pp. 651–657. Ball, D., 1964. Loss-on-ignition as an estimate of organic matter and organic carbon in non-calcareous soils. Journal of Soil Science 15, 84–92. Boswell, E., Koide, R., Shumway, D., 1998. Winter wheat cover cropping, VA mycorrhizal fungi and maize growth and yield. Agriculture, Ecosystems and Environment 67, 55–65. Clark, A., 2007. Managing Cover Crops Profitably, 2nd ed. Sustainable Agriculture Network, Beltsville, MD, USA. Driver, J.D., Holben, W.E., Rillig, M.C., 2005. Characterization of glomalin as a hyphal wall component of arbuscular mycorrhizal fungi. Soil Biology and Biochemistry 37, 101–106. Elliott, E.T., Coleman, D.C., 1988. Let the soil work for us. Ecological Bulletin 39, 23–32. Fore, S.R., Porter, P., Lazarus, W., 2011. Net energy balance of small-scale on-farm biodiesel production from canola and soybean. Biomass and Bioenergy 35, 2234–2244.

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