Non-food lignocellulosic crops with both high biomass yields and superior adaptation to marginal lands have significant potential as biofuel feedstocks that can replace fossil fuels. Deployment of dedicated crops into single biofuels, however, has been reduced by conversion technology costs and low petroleum prices. Integrated biorefinery strategies, in which value-added coproducts are generated in conjunction with biofuels, by comparison offer opportunities to overcome this economic disadvantage. The objective of this research was to evaluate succinic acid accumulation across candidate lignocellulosic feedstocks. Feedstock entries included pearl millet x napiergrass hybrids (“PMN”; Pennisetum glaucum [L.] R. Br. × P. purpureum Schumach.), napiergrass ( P. purpureum Schumach.), annual sorghum ( Sorghum bicolor [L.] Moench), pearl millet ( P. glaucum [L.] R. Br.), perennial sorghum ( Sorghum spp.), switchgrass ( Panicum virgatum L.), sunn hemp ( Crotalaria juncea L.), giant miscanthus ( Miscanthus × giganteus J. M. Greef & Deuter) and energy cane ( Saccharum spp. L.). Replicated field plots, as well as an independent greenhouse trial, were characterized for succinic acid content. The PMN, napiergrass, sunn hemp and energy cane entries had greater ( P ≤ 0.05) succinic acid yields, up to 556 kg·ha-1, in field trials. Napiergrass and PMN entries similarly had higher succinic acid yields under greenhouse conditions; however, irrigation treatments did not alter succinic acid accumulation in this study. Napiergrass, PMN, and energy cane thus are promising biorefinery feedstocks.
Succinic acid is among the Department of Energy’s top value-added chemicals from biomass [
Napiergrass (Pennisetum purpureum Schumach.) is a high biomass perennial that is used primarily as a forage grass throughout the tropical and subtropical regions of the world [
Pearl millet (Pennisetum glaucum [L.] R. Br.) is an annual diploid (2n = 2x = 14). It originated in northern Africa in an area extending from western Sudan to Senegal [
Napiergrass (2n = 4x = 28) can be crossed with pearl millet (Pennisetum glaucum [L.] R. Br.) (2n = 2x = 14) to produce interspecific triploid hybrids (2n = 3x = 21) that have the chemical composition of pearl millet and biomass yield potential of napiergrass [
Switchgrass (Panicum virgatum L.) is a perennial grass indigenous to North America. Switchgrass can be utilized as a forage crop, either grazed or harvested for silage and hay [
Giant miscanthus (Miscanthus × giganteus J. M. Greef & Deuter) is a perennial C4 grass native to Asia. It is a triploid interspecific hybrid between M. sinensis Andersson (a diploid species) and M. sacchariflorus (Maxim.) Hack (a tetraploid species) [
Sugarcane (Saccharum L. spp.) is a perennial grass cultured mainly for sucrose production [
Sorghum is an important crop used mainly for grain and forage production, and it is now being evaluated as a bioenergy crop [
Sunn hemp (Crotalaria juncea L.) is a legume native to India and is used as a soil-improving crop, a green manure, and as livestock feed [
Twelve feedstock entries spanning seven grass species and one legume species were included in both field and greenhouse trials (
In May 2016, replicated plots (n = 3) were planted in a completely randomized design in College Station, Beeville, and Stephenville, TX. The College Station location (30˚32'N, 96˚26'W; elevation 81 m) was on a Weswood silty clay loam (pH 8.0). The Beeville location (28˚27'N, 97˚42'W; elevation 70 m) was on a Parrita sandy clay loam (pH 7.2). The Stephenville location (34˚17'N, 96˚12'W; elevation 370 m) was on a Windthorst fine sandy loam (pH 6.8). The Beeville, College Station, and Stephenville sites were in the United States Department of Agriculture plant hardiness zones 9 a, 8 b, and 8 a respectively. The growing season included 2538, 2858, and 2460 Growing Degree Days (GDD; ˚C), as well as 602, 1105, and 655 mm of total water inputs (irrigation plus precipitation) at the respective Beeville, College Station, and Stephenville locations. Each cultivar
Species | Identification |
---|---|
Pearl Millet-Napiergrass (PMN) | PMN10TX13 |
Napiergrass | Merkeron |
Napiergrass | PEPU 09FL03 |
Napiergrass | PEPU 09FL01 |
BMR sorghum | SDH2942 |
Annual sorghum | SX-17 |
BMR Pearl millet | Exceed |
Perennial sorghum | PSH 09TX15 |
Switchgrass | Alamo |
Sunn hemp | Tropical Isle |
Giant miscanthus | (Mxg) |
Energy cane | (Unknown accession) |
was planted in three plots (3 m × 3 m) with four, 3-m rows. Random green leaf samples collected from field trials at two dates (September and November 2016) were used for succinic acid content assays. Leaf samples were packed in dry ice before being frozen and were kept frozen (−20˚C) until analyzed. Measurement of intracellular concentrations of succinic acid [
To evaluate the drought induction of succinic acid accumulation, all entries were planted in a greenhouse trial in May 2016. Replicated 11L pots (n = 3) were planted in completely randomized design. A potting media mix (Sunshine Redi-Earth Plug & Seed Potting Mix) was used. Plants were maintained in the greenhouse for 3 mo before drought treatments were imposed to allow acclimation of plants to greenhouse conditions. During this period, greenhouse temperatures were kept below 38˚C, no supplemental lighting was utilized, plants were monitored daily and watered as required to maintain field capacity, and no supplemental fertilization applications were made. Treatments included a well-irrigated control and deficit irrigation regime, which were initiated in August 2016. Soil volumetric water contents (SWC) were measured daily using a soil moisture meter (FieldScout TDR 100; Spectrum Technologies, Aurora IL, USA). Well-irrigated plants were watered to maintain soil water content at field capacity. In the drought stress treatment, plants were not irrigated until the volumetric soil moisture content dropped below 15%. Greenhouse temperatures were again maintained below 38˚C, and neither supplemental lighting nor fertilization was incorporated. Leaf samples of plants from each pot were collected on November 15, 2016. Leaf samples were again immediately packed in dry ice upon harvest and kept frozen (−20˚C) until analyzed. Intracellular succinic acid concentrations were quantified using the Succinate (Succinic Acid) Colorimetric Assay kit (Biovision, Milpitas, CA, USA) according to manufacturer’s instructions, and absorbance measurements were made at 450 nm.
The field trial statistical model consisted of location, harvest and plant entry in a three-factorial arrangement looking at three-way interactions and, if those were not significant (P ≤ 0.05), at simple effects. The greenhouse statistical model consisted of irrigation level and plant entry in a two-factorial arrangement looking at two-way interactions and, if those were not significant, at simple effects. Data collected was submitted to analysis of variance and, where appropriate, multiple means separated using All Pair, Tukey’s HSD with JMP software (JMP Pro12, Statistical Analysis System, Cary NC, USA). Differences were considered significant at P ≤ 0.05.
In the field trials, succinic acid concentrations did not vary across sampling dates (
In the greenhouse trial, succinic acid concentrations did not vary across irrigation treatments. However, feedstock entries differed in their response to irrigation levels. PMN and Merkeron had the highest succinic acid concentration under deficit irrigation, while the non-BMR annual sorghum had the lowest succinic acid concentration (
Succinate | ||
---|---|---|
locY | ***Z | |
timeX | ns | |
trtW | *** | |
loc* time | *** | |
loc* trt | *** | |
time* trt | *** | |
loc* time* trt | *** |
ZNS (nonsignificant) or significant at P ≤ 0.05 (*), 0.01 (**), or 0.001 (***). YThree locations. XTwo harvesting time. WPlant entries.
Entry | Succinic acid (g・kg−1) | |||||
---|---|---|---|---|---|---|
Stephenville | College Station | Beeville | ||||
Sept | Nov | Sept | Nov | Sept | Nov | |
PMN 10TX13 | 0.3 cZ | 7.3 a | 4.3 abc | 5.1 a | 11.3 a | 8.0 a |
Merkeron | 3.8 bc | 8.3 a | 5.2 a | 5.4 a | 10.9 a | 7.6 a |
PEPU 09FL03 | 7.4 a | 8.2 a | 4.7 ab | 5.9 a | 12.0 a | 9.3 a |
PEPU 09FL01 | 8.4 a | 8.5 a | 4.3 abc | 5.6 a | 11.1 a | 9.0 a |
BMR Sorghum | 6.1 ab | 5.6 a | 1.9 c | 4.5 a | 11.8 a | 6.7 a |
SX-17 | 1.6 c | 6.9 a | 3.1 abc | 3.8 a | 10.1 a | 7.7 a |
BMR pearl millet | 8.8 a | 6.4 a | 5.5 a | 4.0 a | 10.7 a | 7.3 a |
PSH 09TX15 | 0.5 c | 8.0 a | 2.8 abc | 3.5 a | 12.2 a | 9.0 a |
Alamo switchgrass | 6.3 ab | 7.3 a | 3.4 abc | 4.9 a | 12.0 a | 8.9 a |
Tropical isle Sunn Hemp | 6 ab | 5.7 a | 1.7 bc | 2.5 a | 9.3 ab | 8.3 a |
Giant miscanthus (Mxg) | 0.3 c | 5.6 a | 3.2 abc | 3.6 a | 2.6 b | 6.3 a |
Energy cane | 6.1 ab | 7.2 a | 3.9 abc | 3.7 a | 8.1 ab | 6.1 a |
Z Means within a column under each main factor followed by the same letter are not significantly different according to All Pairs (P > 0.05), Tukey HSD.
Entry | Means | |||
---|---|---|---|---|
Stephenville | College Station | Beeville | ||
PMN 10TX13 | 236 aZ | 370 a | 556 a | |
Merkeron | 188 ab | 77 b | 145 b | |
PEPU 09FL03 | 271 a | 65 b | 143 b | |
PEPU 09FL01 | 153 ab | 33 b | 116 b | |
BMR sorghum | 74 ab | 13 b | 44 b | |
SX-17 | 111 ab | 17 b | 99 b | |
BMR pearl millet | 53 ab | 3 b | 3 b | |
PSH 09TX15 | 84 ab | 13 b | 89 b | |
Alamo switchgrass | 87 ab | 29 b | 55 b | |
Tropical Isle Sunn Hemp | 198 ab | 81 b | - | |
Giant miscanthus (Mxg) | 24 b | 8 b | 12 b | |
Energy cane | 248 a | 50 b | 86 b |
Z Means within a column under each main factor followed by the same letter are not significantly different according to All Pairs (P > 0.05), Tukey HSD.
Entry | Succinate (g・kg−1) | |
---|---|---|
Deficit irrigation | Non-deficit irrigation | |
PMN 10TX13 | 11.1 aZ | 8.0 ab |
Merkeron | 10.1 a | 9.9 a |
PEPU 09FL03 | 9.4 ab | 11.1 a |
PEPU 09FL01 | 9.3 ab | 10.9 a |
BMR Sorghum | 9.5 ab | 0.8 b |
SX-17 | 1.5 c | 9.0 ab |
BMR pearl millet | 10.2 abc | |
PSH 09TX15 | 3.1 bc | 6.9 ab |
Alamo switchgrass | 8.2 abc | 10.4 a |
Tropical isle Sunn Hemp | 4.6 abc | 6.9 ab |
Giant miscanthus (Mxg) | 4.5 abc | 5.3 ab |
Energy cane | 4.3 abc | 7.8 ab |
Grand mean | 7.1 | 7.9 |
Z Means within a column under each main factor followed by the same letter are not significantly different according to All Pairs (P > 0.05), Tukey HSD.
The greatest succinic acid yields were recovered from PMN, napiergrass, sunn hemp and energy cane. Our results demonstrated that biomass yield for feedstocks had a much greater effect on total succinic acid yield than succinic acid concentration within feedstocks. Economic profitability of biofuel strategies can therefore be improved when candidate feedstocks are processed through integrated biorefineries including both primary biofuels as well as succinic acid. A conceptual biorefinery incorporating succinic acid could include the following stages: 1) succinic acid extracted from the liquid fraction prior to ethanol fermentation, 2) bioethanol conversion from cellulose and hemicellulose fractions, 3) biopower from the residual lignin fraction, and 4) mineral bioproducts (biosilica, plant nutrients) from the remaining ash fraction (
The authors declare no conflicts of interest regarding the publication of this paper.
Xu, Y.F., Foster, J.L., Muir, J.P., Burson, B.L. and Jessup, R.W. (2018) Succinic Acid Production across Candidate Lignocellulosic Biorefinery Feedstocks. American Journal of Plant Sciences, 9, 2141-2153. https://doi.org/10.4236/ajps.2018.911155