Research Article pubs.acs.org/journal/ascecg
Electrochemical Evaluation of Sweet Sorghum Fermentable Sugar Bioenergy Feedstock Minori Uchimiya,*,† Joseph E. Knoll,‡ and Karen R. Harris-Shultz‡ †
USDA-ARS Southern Regional Research Center, 1100 Robert E. Lee Boulevard, New Orleans, Louisiana 70124, United States USDA-ARS Crop Genetics and Breeding Research Unit, 115 Coastal Way, Tifton, Georgia 31793, United States
‡
S Supporting Information *
ABSTRACT: Although sweet sorghum is a promising feedstock for bioenergy and biobased products, sweet sorghum-based biorefineries in the U.S. are still in the planning or pilot-scale stages. Accurate, rapid, and inexpensive metrology is known to streamline (bio)refining operations and drive the return on investment. In this study, new cyclic voltammetry (CV)-based methods were developed to rapidly classify sweet sorghum fermentable sugar feedstocks for electroactive functionalities. In addition to providing industrial QA/QC protocols, developed methods could be used to screen for the pest-resistant cultivars containing redoxactive antifeedants (e.g., flavonoids, alkaloids, and aconitic acid), enabling germplasm development for a sustainable feedstock supply chain. Developed CV methods were tested on five male (Atlas, Chinese, Dale, Isidomba, and N98) and three female (N109B, N110B, and N111B) inbred lines and their hybrids (23 cultivars total) planted in April, May, and June of 2015 in Georgia, and harvested at the hard-dough stage. The peak anodic potential (Epa in volts) of derivative CV (pH 5, 0.1 M KCl) overlapped with quercetin and tannic acid model reductants. Fluorescent porphyrin/chlorophyll-like condensed and recalcitrant aromatic structure is likely to be the primary electronenriched (highest CV peak areas) secondary product, and showed significant (p < 0.05) cultivar and planting date dependencies. KEYWORDS: Bioethanol, Biobutanol, Advanced biofuels, Breeding, Organic acids
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INTRODUCTION Sorghum (Sorghum bicolor (L.) Moench) is a heat- and drought-tolerant crop that has promise to supplement corn (Zea mays L.) for biofuel production from fermentable sugars (for sweet cultivars) and lignocellulosic biomass. Sweet sorghum cultivars are defined by juicy stalks enriched predominantly with sucrose and variable levels of glucose and fructose.1 A few largescale sweet sorghum biofuel plant projects have emerged in the U.S. in recent years. However, sweet sorghum in the U.S. is currently only grown on small acreages to make distilled spirits (whiskey, rum) and food products (sweeteners, syrup) for niche markets. Accurate, rapid, and inexpensive metrology is on demand to streamline the (bio)refining operations, to expedite the breeding efforts, and to ensure sustainable feedstock supply chain.2 Cyclic voltammetry (CV) is a powerful electrochemical technique to identify the redox-active components3 in raw and refined agricultural products including grain sorghum,4 cane/ palm sugar,5 and wine.6 Current response (as a function of voltage) is controlled by the diffusion of electroactive species to the electrode surface, and the electron transfer reaction kinetics. The extent and reversibility of both processes depend upon the solution composition and CV parameters including the scan rate.7 When the redox reaction in the anodic direction is chemically irreversible and forms decomposition products, the This article not subject to U.S. Copyright. Published 2017 by the American Chemical Society
return current (in cathodic direction) becomes asymmetrical or absent, indicating the absence of, or different mechanisms of, oxidation involving the byproducts.3 Screen printed electrodes (SPE) have become widely available to develop (1) sensors after the surface modifications, and (2) inexpensive yet accurate methods to detect redox-active species8 on the bare SPE to evaluate the quality of agricultural commodity and bioenergy feedstocks.9 Unlike the traditional glassy carbon electrodes, the SPE surface does not need to be regenerated to remove oxidation byproducts, because it is single-use and disposable.10 On the other hand, sluggish heterogeneous kinetics, unknown surface area of the electrode, and background noise from the carbon ink are the disadvantages of SPE over glassy carbon electrodes. These limitations could be overcome by taking the derivative11 or semi-integral3 to enhance the CV peaks and to extract the electrochemical parameters including the peak anodic potential (Epa in volts). Because no prior reports employed CV to investigate sweet sorghum juice, the present study first developed methods by utilizing the literature sources investigating other agricultural products. Square wave voltammetry and CV of algae extracts Received: May 26, 2017 Revised: June 28, 2017 Published: July 5, 2017 7352
DOI: 10.1021/acssuschemeng.7b01662 ACS Sustainable Chem. Eng. 2017, 5, 7352−7364
ACS Sustainable Chemistry & Engineering
Research Article
(ethanol−water) showed a correlation with 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Folin−Ciocalteu assays for radicals.9 The peak potential of sorghum grain extracts originated from proanthocyanidins/condensed tannin (a subclass of flavonoids),12 and decreased as a function of pH, although the authors did not present the cyclic voltammograms.4 Extensive CV-based classification studies on wine identified the following electroactive components, based on the deconvolution of anodic peaks: anthocyanins, catechins, cinnamic acid derivatives, gallic acid, and other phenolic acids.6 On the basis of the integrated area and the frequency of deconvoluted peaks in wine samples, “redox spectra of wine” was constructed to represent the concentrations of individual phenolics (having unique Epa) to classify wine by the degree of electron-donating capacity.6 Our previous report in this series analyzed the stem juice composition of 23 inbred and hybrid sweet sorghum cultivars planted in April, May, and June of 2015 in southeastern U.S. and harvested at the hard-dough stage.13 Later planting consistently (p < 0.05) (1) increased sucrose, total sugar, and trans-aconitic acid concentrations, Brix, and total organic carbon (TOC), and (2) decreased electric conductivity (EC). Sucrose, total sugar, pH, EC, and Brix showed significant cultivar × planting date interactions.13 Fluorescence excitation emission spectrophotometry with parallel factor analysis (EEM/PARAFAC) identified a conjugated, aromatic, polyphenol-like fingerprint in juice that is expected to be redoxactive.13 The present study was carried out to first screen sweet sorghum juice for redox-active structures by CV. Voltammograms of juice were compared to reference reductants (quercetin, catechin, tannic acid, hydroquinone, gentisic acid, and trolox) covering a range of standard reduction potentials (E0), pKa, and reversibility.3 After identifying the primary contributors to the redox reactivity of juice, Epa and integrated peak area were used to classify the cultivar and planting date effects.
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Figure 1. Raw anodic current (after background subtraction and first derivative with smoothing) of representative juice (points in a) and methanol (bagasse) or ethanol (leaf, panicle, and lignin, all 20 g L−1) extracts of sweet sorghum biomass (lines in b−d) and lignin (e). In (a), Gaussian fits are given as lines. All CVs were obtained at pH 5 (40 mM phosphate buffer) in 0.1 M KCl (b−e contained 50% solvent).
MATERIALS AND METHODS
Field Experiment. As described previously,13,14 the field experiment was conducted near Tifton, GA (31° 29′ N, 83° 31′ W) on a Tifton loamy sand (fine-loamy, kaolinitic, thermic Plinthic Kandiudults). Three inbred lines (N109B, N110B, and N111B)15 were designated as females, and five inbred lines (Atlas, Chinese, Dale,16 Isidomba, and N98)15 were designated as males. The male-sterile (A-line) versions of the female lines were used as seed parents to generate hybrids, while the male-fertile (B-line) versions were planted in this study. All 15 possible hybrids were generated, for a total of 23 entries (Table S1). In 2015, all 23 entries were planted in a randomized complete split-plot design with three replications. Planting date was used as the main plot factor and entry as the split-plot factor. Planting dates were April 22 (planting 1), May 14 (planting 2), and June 16 (planting 3). Each plot consisted of two 6 m-length rows, 0.9 m apart. Granular 10-10-10 fertilizer was applied prior to planting at a rate of 560 kg ha−1, followed by a side-dress liquid application of 112 kg ha−1 N at approximately one month after planting. Weeds were controlled by applying pendimethalin (N-(1-ethylpropyl)-3,4dimethyl-2,6-dinitrobenzenamine), atrazine (1-chloro-3-ethylamino-5isopropylamino-2,4,6-triazine), and bentazon (3-isopropyl-1H-2,1,3benzothiadiazin-4(3H)-one 2,2-dioxide), all at the manufacturers’ recommended rates. Irrigation was applied only as needed to speed germination, and no insecticides were applied. Harvest dates were set to the hard-dough stage of maturity (when Brix typically peaks), and ranged from July 23, July 30, August 6, to August 13 for planting 1; August 13, August 20, to August 27 for planting 2; and September 9, 17, to September 24 for planting 3. Three representative stalks were harvested from each plot, panicles and leaves were removed, and juice was extracted from the stems by
passing twice through a portable three-roller mill (Sor-Cane PortaPress, McClune, Reynolds, GA). Juice samples were immediately frozen after measuring the soluble solids concentration (Brix) using a digital refractometer (Refracto 30GS, Mettler-Toledo, Columbus, OH).14 The leaf, bagasse, and panicle portions were dried at 60 °C until the weight stabilized, ground in a Wiley mill (Thomas Scientific, Swedesboro, NJ), and sieved (
