Novel storage technologies for raw and clarified syrup biomassfeedstocks from sweet sorghum (Sorghum bicolor L. Moench)
Gillian Eggleston a, *, Brett Andrzejewski a, Marsha Cole a, Caleb Dalley b, Scottie Sklanka a,Eldwin St Cyr a, Yoo-Jin Chung a, Randall Powell c
a USDA-ARS Southern Regional Research Center, 1100 Robert E. Lee Blvd., New Orleans, LA 70124, USAb USDA-ARS Sugarcane Unit Houma, LA 70360, USAc Delta BioRenewables, LLC, Memphis, TN, USA
a r t i c l e i n f o
Article history:Received 19 February 2015Received in revised form2 July 2015Accepted 12 July 2015Available online 6 August 2015
Abbreviations: Rt, retention time; MOL, milk of limeter turbidity units; CJ, clarified juice; IC-IPAD, ion chrpulsed amperometric detection; HPLC-RI, high performwith refractive index detection; GL, green leaves; BFood and Drug administration e generally recognizedparticulate absorption.* Corresponding author.
Attention is currently focused on developing sustainable supply chains of sugar biomass feedstocks fornew, flexible biorefineries. Fundamental processing needs identified by industry for the large-scalemanufacture of biofuels and bioproducts from sweet sorghum (Sorghum bicolor L. Moench) includestabilization and concentration of juice into syrup for long-term storage, year-round supply, efficienttransport, and acceptable end-product yields. Pilot plant studies were conducted to evaluate the storage(up to 160 days at ~25 �C) of raw and clarified syrups from sweet sorghum hybrid and commercialcultivars. Clarified syrups were manufactured after clarification of juice (80 �C; 5 ppm polyanionicflocculant) at various target limed pHs (6.1e6.8) followed by vacuum evaporation. All 70 Brix raw syrupswere susceptible to microbial deterioration on the surface during storage, and raw syrups were moresusceptible than clarified syrups. Surface deterioration was mainly fungal since bacterial growth wasinhibited by low water activity. Juice clarification reduced the loss of fermentable sugars during theevaporation stage and, generally, allowed for better storage of syrup up to 80 days. Target clarification pHhad a dramatic effect on the storage of clarified 70 Brix syrups with more acidic pHs reducing fungaldeterioration. Further studies are now warranted on the post-evaporation pH adjustment of raw andclarified syrups to <6.1 for long-term storage. Inexpensive soy bean oil and candellila wax showedpromise as surface sealants to preserve syrups for at least 80 days of storage at ~25 �C, also warrantingfurther investigation.
Renewable biomass raw materials are assuming increasedimportance for bioenergy and industrial chemical manufacturers.New commercial-scale projects, advances in processing of first andsecond generation feedstocks, and novel catalyst developmentexemplify efforts to extend the role of renewables [1]. While muchresearch has been focused on process optimization for advanced
e (CaOH2); NTU, nephelom-omatography with integratedance liquid chromatographyL, brown leaves; FDA-GRAS,as safe; HEPA, high efficiency
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and second generation biofuels as well as bioproducts, attention isnow focused on developing sustainable supply chains of sugarfeedstocks for the development of new, flexible biorefineries [2].This includes harvest operations, advanced equipment productiv-ity, improved feedstock quality, and cost-effective approaches forminimizing feedstock sugar losses during storage [2]. Sweet sor-ghum has been widely recognized as a promising sugar feedstockcrop due to its efficient C-4 photosynthetic pathway, easy cultiva-tion from seed, ability to grow onmarginal lands with low fertilizerand water requirements, possibility of multiple crops per season,wide geographic suitability across the USA, directly fermentablesugars, and huge breeding potential [3].
A major technical challenge of using sweet sorghum for biofuelsand bioproducts is the fast spoilage of juices [4]. Sweet sorghumjuice is highly susceptible to microbial deterioration and to a lesserextent chemical and enzymatic deterioration [5,6] because of highwater and sugar contents. As a consequence, one of the
G. Eggleston et al. / Biomass and Bioenergy 81 (2015) 424e436 425
fundamental processing areas that need to be addressed for sweetsorghum includes the stabilization and concentration of juice into astable syrup for efficient and effective transport, storage, and year-round supply. Concentrating sweet sorghum juice into syrup im-proves transportation from a central processing point to an end-user (short and long transportation distances). Moreover, someU.S. companies' business plans emphasize the production of syrup.Manufacture of storable, sweet sorghum syrup would also helpoffset the problem that can arise when the crushing capacity isgreater than the distillery capacity. Often, the conversion of thesweet sorghum sugars to value added products is the rate limitingstep in fermentation [7].
The ability to store syrup for a minimum of 4 months has beenrequested by numerous companies. Unfortunately, no informationhas beenpublished on the storage of sweet sorghum syrups, and verylittle on sugarcane syrups. Honig [8] reported Saccharomyces cer-evisiaeyeastoccurred in67Brix (%dissoloved refractometric dissolvedsolids) sugarcane syrups; S. rouxiioccurred in72e76Brixpartial invertsyrups, while Rhodotorula and Candida occurred in 55 Brix syrup.Unfortunately, the syrupqualityand sugarprofileswerenot examinedafter storage. In comparison, largequantities of sugarbeet syrup (thickjuice) are often stored at European and someUS sugar beet factories toextend their sugar campaigns, and there have been some publishedreports [9,10]. The target Brix for the storage of beet syrup is >68 Brix[10] and the storage temperature should be below 20 �C. Unlike beetsugar production in temperate climates, however, sweet sorghumproduction tends to occur inmore semi-tropical and tropical climates(although it is also grown in temperate climates), and storage at 20 �Ccannot be achieved inexpensively.
Sweet sorghum syrups can be produced directly from raw orclarified juices. The goals of juice clarification are to: (i) stabilizejuice with respect tomicrobial deterioration, (ii) remove suspendedand turbid particles, and (iii) allow subsequent concentration of theclarified juice (CJ) into a viable liquid product using standardcommercial evaporation technologies. We recently developed aclarification method in the laboratory for sweet sorghum juices [3],that was validated at the pilot plant scale [4]. Sweet sorghum juiceis first heated to allow colloidal particles, particularly proteins, tocoagulate and form natural flocs. Hydrated lime (calcium hydrox-ide) is then added in the form of milk of lime (MOL), to the heatedsweet sorghum juice to increase the pH to 6.5 for (i) neutralizationof acids, (ii) reduction of unwanted acid degradation of invertsugars in downstream thermal evaporation, (iii) formation of cal-cium phosphate flocs, and (iv) introduction of positively chargedparticles in the juice solution [3]. Calcium, from the juice and MOL,and phosphorous that occurs naturally in the sweet sorghum juiceare necessary for the formation of calcium-phosphate bridges thataid in the flocculation process. Better and faster floc precipitation isfurther aided by HMW polyanionic acrylamide flocculants whichagglomerate particles and add weight.
At ambient temperatures, microbial degradation is the majorcause of syrup spoilage or deterioration [5,12], and different culti-vars of sweet sorghum with varying sugar compositions are ex-pected to maintain different flora of microorganisms. Theobjectives of this study were to evaluate the effect of pilot plantclarification of sweet sorghum juice [11] on syrup production andstorage, and to develop syrup storage technologies that are techno-cost efficient. An optimal syrup storage method will (i) use a min-imum of energy and costly processing aids, (ii) be free of biocidesthat could interfere with end-product fermentation, (iii) preservefermentable sugars and other important macro- and micro-nutrients in the sugar syrup, and (iv) maximize yields of down-stream fermentable sugars.We investigated the storage of both rawand clarified sweet sorghum syrups, taking into account somesuccessful technologies already found for beet syrup storage.
2. Material and methods
2.1. Chemicals
Powdered, hydrated lime (Carmeuse, Pittsburgh, PA, USA) andflocculant (Praestol 2640 z, ~2.7 MDa copolymer of acrylamide andsodium acrylate with a medium charge density [Stockhausen,Krefeld, Germany]) were kindly provided by the staff at LafourcheSugars, LLC (Thibodaux, LA, USA). Flocculant (1 g) was added to 1 Lof deionized water and allowed to thoroughly mix for 1 h and sit for24 h before use. Milk of lime (MOL) was prepared by adding hy-drated lime (120 g) to 1 L of deionized water, and stirred vigorouslybefore use. Analytical grade hydrochloric acid, sodium hydroxide,triethanolamine, 1-amino-2-napthol-4-sulfonic acid and Candelillawax were purchased from SigmaeAldrich (St. Louis, MO, USA), andsodium chloride and magnesium chloride from J. T. Baker (CenterValley, PA, USA). Sodium acetate trihydrate came from Fisher Sci-entific (Fair Lawn, NJ, USA). Soybean oil (Crisco, Orrville, OH, USA)was purchased from a local grocery store. Rock salt was purchasedfrom Lowes (New Orleans, LA).
2.2. Sweet sorghum production and juice extraction
2.2.1. 2011 Syrup study in Tennessee (TN)In 2011, sweet sorghum juice was provided by Delta Renew-
ables, Memphis, TN. Specifically, juice was obtained from sweetsorghum Hybrid-A on Sept 14, 2011 after planting on June 1, 2011,commercial cultivar M81E on Oct 20, 2011 (planted on July 8) andNov 10, 2011 (planted on June 15). Sweet sorghum billets (~25 cmlength) were harvested with a Case New Holland (Burr Ridge, IL)sugarcane combine harvester. The top seed heads were removed bythe top-cutter on the harvester. The sweet sorghum billets werecrushed by passing through a custom built 4-roll mill (Jefferson-ville, KY, USA) followed by a Laurel Machine and Foundry Co(Laurel, MS, USA) 3-roll mill. The combined first and secondexpressed juice was mixed and filtered through a 0.6 mm pore sizescreen and stored in three 114 L drums. Biocide (~150 cm3; Busan881™, Buckman Labs., USA) was added to each of the three drumscontaining juice on each sampling date. The juice was transportedin a cargo tote (1.2 � 1.0 � 1.2 m) containing a salt (sodiumchloride)-water ice bath, with ice and salt added as necessary tomaintain a temperature below 6 �C during transport (~6 h) to theUSDA-ARS-Southern Regional Research Center (SRRC) sugar pro-cessing pilot plant in New Orleans, LA. At the pilot plant, 70 Brixraw and clarified syrups were produced as described in Section 2.3.The target limed pH for clarification was 6.3 for all three sampledates. The 70 Brix syrups were stored outside for 0e160 days (seeSection 2.4 for details).
2.2.2. 2012 Syrup studies in Louisiana (LA) and Tennessee (TN)2.2.2.1. Houma, Louisiana. Two commercial sweet sorghum culti-vars (Dale and Theis) were planted on April 20, 2012 at the USDA-ARS Sugarcane Research Unit in Houma, LA. Plots consisted of asingle raised bed row measuring 1.8 m wide and were at least107 m in length with four replicates. Two rows of sorghum with a60 cm spacing were planted on top of each bed at a seeding arealdensity of ha�1. Plots were fertilized as normal for sorghum(90 kg ha�1 N, 22 kg ha�1, K, 45 kg ha�1 P) and treated withmetolachlor plus atrazine (1.42 plus 1.2 kg ha�1) at planting fol-lowed by pendimethalin 1.6 kg ha�1) upon reaching the four leafgrowth stage. Seeds were treated with safener fluxofenim(0.4 g kg�1 seed), which allowed for the metolachlor application.Cultivar Dale was first harvested to investigate the effect of leaveson processing performance and syrup storage ability. On Aug 8,2012, multiple whole-stalks of Dale were harvested by hand,
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topped, and stripped of all leaves, and 265 L of juice extractedthrough a Squier™ (Buffalo, NY, USA) three-roller mill at ArdoyneFarm (Houma, LA) with imbibition water (~15% mass ratio of wateradded to the fresh weight of stalks) on the second pass. The juicewas filtered through a 0. 6 mm mesh filter. Raw juice was imme-diately transported (~1 h) in drums chilled in icy-salt water to theUSDA-ARS-SRRC pilot plant in New Orleans where a portion of thejuice was clarified, as described in Section 2.3. The limed juicetarget pHwas 6.5 as the 2011 syrup experiments had indicated a pHof 6.3 resulted in the acid degradation of fermentable sugars duringclarification and subsequent evaporation [11]. Raw and clarifiedjuices were then evaporated into raw and clarified syrups to both70 and 80 Brix levels in the pilot plant. On the next day Aug 9, 2012,twenty-eight bundles (twenty-five whole-stalks per bundle) ofDale were harvested by hand and topped, but the leaves were notremoved. Juice was similarly extracted, transported, and processedinto raw and clarified syrups (70 and 80 Brix) as the Aug 8 samples.Random field stalks of Dalewere also similarly separated into stalk,brown (BL) and green leaf (GL) tissues and the fresh material massfractions of the stalk, GL, and BL were 81.2, 16.8, and 2.0% BL,respectively. GL clung tightly to the Dale stalk and white wax wasvisible especially on the lower internodes. Secondary or auxiliaryseed heads were visible.
The second sweet sorghum cultivar, Theis, was hand-harvestedin Houma, LA, and the leaves were not stripped before juiceextraction. Juice extraction, transportation, and processing into rawand clarified syrups (70 and 80 Brix) was similarly performed as forDale but the effect of different limed juice target pHs was investi-gated (pHs 6.1, 6.5, and 6.8) with a separate target limed pH studiedeach day (Aug 21e25, 2012). Twenty-five random field stalks ofTheis were also separated into stalk, BL, and GL and the fresh ma-terial mass fractions were 89.2, 7.3, and 3.5%, respectively. GL clungtightly to the Theis stalk but little wax was visible. Secondary orauxiliary seed heads were visible.
The raw and clarified syrups from Dale and Theis were subse-quently subjected to storage experiments over a 160 day storageperiod as described in Section 2.4.1.
2.2.2.2. Memphis, Tennessee. In 2012, sweet sorghum juice wasprovided by Delta BioRenewables LLC at their new biorefinery sitein central Memphis, TN,M81E cultivar (planting date May 22, 2012)was harvested into ~25 cm billets on Oct 1, using a Case NewHolland (Burr Ridge, IL) sugarcane combine harvester with the topcutter on. Also on Oct 1, twenty-five random field stalks of M81Ewere hand-cut and separated into stalk, BL and GL, and seed heads(sometimes auxiliary seed heads were present), and the freshmaterial mass fractions were calculated. In the early morning of Oct2, three random samples (~6.8 kg each) were collected from the pileof combinedM81E billets and separated into stalk, green and brownleaves, and seed heads to determine how much sweet sorghumextraneous matter was blown off by the combine harvester fans.Later in the morning of Oct 2, juice was extracted from the billetedcane by extracting it twice across a three-roller sorghum mill, andthen filtered as described in Section 2.2.1. The filtered juice wasadded into three 114 L drums with biocide (~150 cm3; Busan 881™,Buckman Labs., USA) added and similarly transported to NewOrleans as in 2011. On arrival to the pilot plant,151 L of the juicewasimmediately clarified and the target limed juice pH was 6.5. Whileclarification occurred, the raw (non-clarified) juice was evaporatedinto 70 and 80 Brix syrups. Once the clarified juice (CJ) was ob-tained, it was also immediately evaporated into 70 and 80 Brixsyrups. Samples across both clarification and evaporator processeswere collected and stored in a �40 �C freezer until analyses.
Sampling was repeated on Oct 30, 2012 with Dale sweet sor-ghum cultivar (planting date June 20, 2012) and on Nov 27 with
M81E sweet sorghum (planting date May 22, 2012) and similarlytreated as theM81E sweet sorghum on Oct 1. Tissue components onwhole-stalk and billet samples were also similarly measured. On 12and 27 Nov, only 80 Brix syrups were produced. On Nov 27 theM81E field sweet sorghum had mostly brown leaves due to recentcold weather.
2.3. Pilot plant manufacture of raw and clarified syrups
2.3.1. Raw syrupSweet sorghum raw juice was processed directly into raw syrup,
on the same day of harvest and transport, in the USDA-ARS-SRRCpilot plant “mini” evaporator unit (210 L full capacity) operatedunder vacuum, which is based on a Robert's type calandria (risingfilm) evaporator frequently operated in sugarcane factories (for fulldetails on design and operation see Ref. [13]). Raw juice was fedinto the evaporator at the bottom by suction until it covered justabove the calandria tubes (30.3 L). The raw juice was then boiledunder ~51 kPa at ~82.2 �C to ensure no orminimal entrainment. TheBrix was allowed to steadily increase and boiling continued underthese conditions until the syrupwas ~45 Brix. The vacuumwas thenincreased to 67.7 KPa and the temperature was lowered to ~74 �C tosimulate multiple-effect evaporators. Eventually the vacuumpressure was increased to 85 kPa at 60e66 �C with the raw juicecontinuously fed into the evaporator until 80 Brix was reached, andthen dropped out of the evaporator at the bottom. The syrup wasseparated into two halves with half kept at 80 Brix while the otherhalf was diluted with de-ionized water to 70 Brix.
2.3.2. Clarified syrupFor clarified syrup production the sweet sorghum raw juice was
first clarified into CJ in the USDA-ARS-SRRC pilot plant [13], and theCJ was then evaporated into clarified syrup the same as for rawsyrup production described above. Raw juice (~151 L) was me-chanically pumped into a 265 L juice tank where the juice washeated (~274 K min�1 or 1.3 �C min�1) to 80 �C with 69 kPa steamand constant stirring. Temperature and pH of juice inside the tankweremonitored using sensors.When the juice reached 80 �C, it wasimmediately limed to the desired target pH with MOL undercontinuous mixing. Once the target pH was obtained the mixer wasturned off and flocculant (Stockhausen™ polyanionic solution;5 mg L�1) was added and stirred by hand using a paddle for 1 min.The flocculated, heated limed juice (FHLJ) was then immediatelygravity fed into a settling or clarification tank below and allowed tosettle. The rate of settling was monitored via test port valves on theside of the clarification tank [13]. A sample was collected in a test-tube at specified time intervals and settling visually observed infront of a bright lamp. Settling time was taken as the time for mudto settle to 25% volume capacity of the settling tank, i.e., at the #6settling port valve. Mud was gravity drained from the bottom valveand stored in a�40 �C freezer until analyzed. The CJ formed (~114 L)was fed into stainless steel holding tanks (19 L) and stored over-night in a walk-in cooler at 4 �C. The next morning the CJ wasevaporated under the same conditions as the raw juice (see Section2.3.1).
2.4. Syrup storage experiments
Syrups produced in the pilot plant were separated into aliquotsand poured into 19 L buckets. Initial studies were performed instainless steel buckets. Stainless steel is often the storage vessel ofchoice, but is muchmore expensive than polymeric materials. Thus,food grade high density polyethylene (HDPE) buckets were used tostore subsequent sweet sorghum syrups. Re-used buckets werethoroughly cleaned and sterilized with ethanol (70% volume
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fraction) and allowed to air dry. All samples stored in buckets weretightly covered with lids. A minimum of 4.2 L of syrup were placedin each stored bucket. All syrups were sampled after 0, 1, 2, 5, 10, 20,40, 80, and 160 days by removing ~40 cm3 from each bucket afterthorough mixing. Before sample removal, the surfaces of the storedsweet sorghum were photographed with a digital still camera (5.1megapixels) on days 80 and 160. All syrup samples were frozen(�40 �C) until analyzed.
2.4.1. The effect of initial 70 and 80 Brix levels on syrup storageDiscussions with staff of Delta BioRenewables indicated that
industry would prefer to store syrups in large tanks outside theirprocessing facilities, similar to the sugar beet industry. Thus, both70 and 80 Brix Dale and Theis raw and clarified syrups fromHouma,LA (see Section 2.2.2), were stored outside the USDA-ARS-SRRCfacility in a shaded area at ambient temperature. Daily high, low,and mean temperatures were recorded from the official weatherstation KNEW (New Orleans, Lakefront airport, LA) nearby [14].Temperatures of the syrups were checked periodically to verify theofficial temperatures matched the stored syrup temperatures,negating any microclimate temperature effects.
2.4.2. The effect of surface oil on syrup storageM81E raw and clarified syrups fromMemphis, TN, at both 70 and
80 Brix levels (see Section 2.2.2) were stored with and without(control) a layer of inexpensive soybean oil on the surface. Soybeanoil (500 cm3) was added to the top of the stored syrups in buckets(19 L) to create a surface oil layer of ~0.85 cmdepth. TheM81E syrupswere stored outdoors in a shaded area at ambient temperature. Rawand clarified 70 and 80 Brix Dale syrups from Memphis, TN, (seeSection 2.2.2) were also stored with and without (control) a layer ofsoybean oil on the surface, in an air conditioned room (25 �C).
2.4.3. The effect of surface wax on syrup storageM81E raw and clarified 80 Brix syrups from Memphis, TN, (see
Section 2.2.2) were stored with and without (control) a layer ofcandellila wax on the surface. Candellila wax (500 g) was meltedover a hot plate and poured on the syrup surface in the bucket tocreate a surface wax layer of ~0.85 cm depth. Aliquot samples weretaken after fracturing the solid wax coating with a glass rod andremoving 40 cm3 of the syrup. The wax layer was then resealedwith some newly melted wax. The M81E syrups were stored bothoutdoors and indoors as described above.
2.5. Brix (percent dissolved refractometric solids), pH, and turbidity
Brix was measured using an Index Instruments (Kissimmee, FL,USA) TCR 15e30 temperature controlled refractometer accurateto ± 0.01 Brix, and results expressed as an average of triplicates.Juice pH was measured on a Metrohm Brinkman 716 DMS Titrino(Riverview, FL, USA) with a Mettler Toledo (Columbus, OH, USA)xerolyte electrode. Syrup pH was measured after the syrup wasdiluted to 18 Brix with de-ionized water and after 3 min of equi-librium at room temperature. Nephelometer turbidity (NTU) mea-surements were taken on a Hach 2100N turbidimeter (Loveland,CO, USA); results are an average of five measurements.
2.6. Sugars and sugar alcohols measured using ion chromatographywith integrated pulsed amperometric detection (IC-IPAD)
Glucose, fructose, sucrose, and mannitol in juice and syrupsamples were determined by IC-IPAD using CarboPac PA1 analyticaland guard columns (Dionex Corp., Sunnyvale, CA, USA) followingthe method reported in Ref. [3]. Results are expressed as g L�1 on a% Brix basis.
2.7. Water activity measurements
Sweet sorghum syrup samples (15 cm3) were placed on anevaporation dish and kept in a laboratory desiccator with a Thermoe hygro sensor (Oregon Scientific, Tualatin, OR, USA) for 24 h.Water activity was calculated from equilibrium relative humidity.The humidity sensor was calibratedwith saturated sodium chlorideand magnesium chloride solutions.
2.8. Organic acids
Organic acids were determined using a Dionex ES50 HPLC sys-tem (Sunnyvale, CA, USA) with refractive index (RI) detection(Shodex RI-101, Yokahama, Japan). Samples were first filteredthrough a 0.45 mmPVDF filter and separated on Phenomenex RezexROA analytical acid and guard columns (Torrance, CA, USA). Organicacids were separated over a 30 min isocratic method with2.5 mmol L�1 sulfuric acid eluent at 1 cm3 min�1, and a columntemperature of 30 �C. Samples were maintained at 4 �C using aDionex AS-1 refrigerated auto-sampler (Sunnyvale, CA, USA).
2.9. Statistics
Harvest and tissue datawas analyzed using PROC GLM in SAS 9.3(SAS Institute, Cary, NC, USA). Means were separated using Dun-can's New Multiple Range Test. Student's t-test, independent, two-tail, was performed to compare the difference of the raw andclarified juice or syrup using Microsoft Excel 2007 (Redmond, WA,USA).
3. Results and Discussion
3.1. Tissue levels before and after combine harvesting
Sweet sorghum biomass tissue components as a fresh materialmass fraction from M81E and Dale cultivars produced at DeltaBioRenewables, TN in 2012 were studied and results are listed inTable 1. From Oct 2 to Nov 28, the stalk component, as a per cent ofthe whole-stalk biomass of M81E, increased from 68.8 to 84.2%,however, the weight per unit stalk varied from 0.49, 0.26, and0.39 kg on Oct 2, Nov 12, and Nov 28, respectively. As expected, thegreen leaves (GL) dramatically decreased from 24.2 to 0.6% onmaturation, whereas the occurrence of brown, senescing leaves(BL) steadily increased from 5.1 to 9.2% over the same time period(Table 1). This was further highlighted with the GL/BL ratiochanging from 4.8 to 0.1 on maturation.
The effect of combine harvesting the field whole-stalks intobillets is also listed in Table 1. The top-cutter of the combineharvester was utilized, thus the amounts of seed heads weredramatically lower in billets than whole-stalks, and the low valuesfor billets in Table 1 were because of the presence of auxiliary seed-heads and possibly from stalks too short to be topped. Typically,there is a small portion of sweet sorghum stalks in the field that areshorter than the majority making removal of all seed headsimpractical. The combine harvester extractor fans removedconsiderable amounts of both GL and BL, but the amount left on thebillets was highly dependent (R2 ¼ 0.77) on the initial amount ofthe respective leaves before combine harvesting (Table 1). The GL/BL ratio was considerably higher in billets than the whole-stalks,mostly due to the lower amount of BL. Eggleston et al. [15] re-ported that in sugarcane BL are more easily blown off than GLbecause of their lower density. Furthermore, sweet sorghum stalkdensities are typically lower than for sugarcane, which will requirecombine harvesting using lower speeds on the extractor fans andthis will lead to higher amounts of leaves remaining.
a Different lower case letters represent statistical differences at the 5% probability level for each tissue in one row only.b The average weight of one stalk of M81E cultivar was 0.49, 0.26, and 0.39 kg on Oct 2, Nov 12, and Nov 28, 2012, respectively.c The average weight of one stalk of Dale cultivar was 0.57 kg on Oct 29, 2012.
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The average weight per stalk of Dale sweet sorghum wasconsistently higher than M81E (Table 1). Considerably less(P < 0.05) BL occurred in Dale than any of the M81E samples atdifferent dates which elevated the GL/BL ratio (Table 1). Similar toM81E, however, the GL/BL ratio was higher in billets than whole-stalk.
3.2. Pilot plant clarification of sweet sorghum juices
Pilot plant clarification performance parameters of the sweetsorghum juices obtained fromDelta BioRenewables, TN in 2011 and2012, and from Houma, LA in 2012, are listed in Table 2. For juicesobtained in 2011, there was 95e98% turbidity removal across clar-ification after only 30e50 min of clarification (Table 2) [11]. Thishigh percentage of turbidity removal may not, however, be neces-sary for extended syrup storage and downstream fermentations[16], and turbidity removal requirements will most likely vary withthe fermentation end-product.
Sweet sorghum juices obtained from cultivar Dale in Houma onAug 8, 2012, had leaves removed from the stalk and were comparedwith leaves remaining on the stalk on Aug, 9, 2012. The presence ofleaves markedly improved the settling time from 45 to 20 min(Table 2), although the turbidity removal and clarified juice (CJ)turbidity values were slightly (P < 0.05) higher than with no leaves(Table 2). The high protein content in leaves would have helped to
Table 2Pilot plant clarification of sweet sorghum juices from Delta BioRenewables, TN Houma, L
form natural flocs on heating during clarification, which wouldhave improved settling [3]. In 70 and 80 Brix clarified syrupsmanufactured from stalks with leaves left on, considerably higheramounts of glucose (up to 27.1e33.9%), fructose (up to 26.5e50.1%)and sucrose (up to 30.5e39.5%) were measured compared to clar-ified syrups manufactured from stalks with leaves removed. Morefermentable sugars represent another advantage of keeping leaveson the stalk. Coble et al. [17] reported that an advantage of leafremoval was higher yields of ethanol, which they stated may havebeen due to lower bacterial load in juices [4]. Lamb et al. [18],however, reported that leaves left on the sweet sorghum stalk,particularly dry BL, absorb juice from the stalk. On a commercialscale, it will be impractical to remove leaves because of undue timeand effort [19]. Increasing the fan speed of the combine harvesterwould only blow out more billets with the leaves which is veryuneconomical [15]. Overall, these results suggest that leaves aid theclarification settling time and may enhance sugar yields, and thedifferences with leaves removed are not great enough to considerremoving more leaves.
Sweet sorghum juices obtained from cultivar Theis in HoumafromAug 21e25, 2012, were subjected to different target limed pHsduring the clarification. As shown in Table 2, the target pH had asignificant (P < 0.05) effect on clarification. The best turbidityremoval was at target pH 6.8 which is most likely attributed to theformation of more calcium-phosphate bridge flocs that aid
A and in 2011 and 2012 (average of three replicates).
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precipitation during clarification [3,11]. Conversely, the worstturbidity removal occurred at pH 6.1 because not enough calcium-phosphate bridged flocs formed. However, at pH 6.8 the addition ofextra calcium from MOL needs to be limited because it may causeundesirable flocculation of certain yeasts. On the other hand, attarget pH 6.1 too much acid degradation of sugars will occur, thus atarget pH 6.5 is optimum. Andrzejewski et al. [11] previously re-ported that pH 6.5 was optimum for pilot plant clarification ofsweet sorghum juice because of reduced losses of sucrose, glucose,and fructose during clarification and subsequent vacuum evapo-ration. It was interesting to note that even at the different targetpHs, turbidity removal, and CJ turbidity values were consistentlybetter for Theis than Dale juices. This is further evidence of a strongcultivar effect on clarification, previously reported by Andrzejewskiet al. [3,11].
Sweet sorghum juices from immature andmature cultivarM81Eas well as mature Dale cultivar obtained from Delta BioRenewables,TN in 2012 were also clarified in the pilot plant to manufacturesyrups for storage studies (Table 2). Again, a strong cultivar effecton clarification performance was observed. The immature M81Ejuice clarified the best, which may be because of the lower Brix ofthe juices [3]. When the juice was not pH adjusted during clarifi-cation, considerably less turbidity removal occurred and
Fig. 1. Photographs of the surface of raw (upper row) and clarified (CJ) (lower row) syrups p(22e26 �C). The dark areas are syrup. Colonies of green, brown, pink, and white fungi are
fermentable sugars were lost due to acid degradation reactions(results not shown). This highlighted the critical need to adjust thejuice pH for processing of juices and syrups.
3.3. Initial storage experiments of raw and clarified syrups fromTennessee, 2011
The first syrup storage experiments were conducted on raw andclarified 70 Brix syrups produced from sweet sorghum juices ob-tained from Delta BioRenewables in 2011. The syrups were storedoutside in a shaded area at ambient temperature, as Delta Bio-Renewables were interested in storing syrup in a large tank outside,protected from the elements. Typical results are shown for syrupstorage after 0e160 days in Figs. 1e3. Both raw and clarified 70 Brixsyrups exhibited some surface microbial deterioration (Fig. 1). Thisbecame worse between storage Days 80e160, although the rawsyrups were more susceptible than clarified syrups especiallyduring the first 80 days. Heterogeneous colonies of green, brown,pink, and white fungi were visible on the surface of the syrups. Ifthe relative humidity in the air is greater than the equilibriumwater activity (Aw) of the syrup, moisture will move from the airinto the syrup surface layer (condensation) increasing the Aw at thesurface. The syrupeair interface with the localized lower water
roduced in 2011, after storage for (A) 80 days and (B) 160 days at ambient temperaturevisible.
Fig. 2. Chromatograms on stored raw syrup from 8 Nov, 2011, at 0 day (black line) and 80 days (blue line) after storage. A. IC-IPAD chromatogram of sugars, sugar alcohols, andoligosaccharides. B. HPLC-RI chromatogram of organic acids. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of thisarticle.)
G. Eggleston et al. / Biomass and Bioenergy 81 (2015) 424e436430
activity was, therefore, where the fungal colonies were found. Thedesign of syrup storage tanks could help to reduce the headspaceand, therefore, surface contact with air and condensation. Dick-erson and Read [20] reported that the geometry of the tank as wellas the volume and viscosity of the syrup will be expected to affectthe rate of cooling of the stored syrup. Filling the storage tank to themaximum will ensure minimum headspace during the storageperiod. Fans have been used in the sugar beet industry (GeoffParkin, Alltech, personal communication) to clear condensation bysweeping the headspace with HEPA filtered air, although HEPAsystems are expensive to maintain. Pumping the syrup into tankswhen the syrup temperature is relatively low will also reducecondensation and remove the surface dilution layer. Frequentmonitoring of stored syrups at sampling points across the height ofthe tank will help to take steps to prevent evenworse deteriorationfrom occurring.
The growth of lactic acid Lactobacilli bacteria did not occurduring storage of the syrups as no increase in the metabolitemannitol was observed [5], as illustrated in Fig. 2A. This was furtherevidenced by the lack of formation of acetic and D-lactic acids, aswell as isomaltose and isomaltotriose [5,21]. In contrast, sweetsorghum juices have been reported to be susceptible mostly toLactobacillus bacterial deterioration, including Leuconostoc mesen-teroides, because of higher available water in juices [12,19,22]. TheM81E clarified syrup, produced on Nov 8, 2011, had the least
microbial growth on its surface after 80 and 160 days of storage(Fig. 1).
Changes in the concentrations of sucrose, glucose, fructose andtotal sugars in the stored syrups are illustrated in Fig. 3. It wasclearly shown that on Day 0, before any storage, the totalfermentable sugars in the syrups were consistently higher in theclarified than raw syrups (Fig. 3). This can be attributed to a muchhigher loss of sugars during the evaporation of raw juice into rawsyrup than evaporation of clarified juice into clarified syrup. Thehigher pHs of the clarified juice will have limited the thermal, aciddegradation of sugars during evaporation [23]. This is an importantresult, as syrup production without pH adjustment will greatlyreduce yields of fermentation end-products.
All the stored 70 Brix syrups lost considerable amounts of totalsugars during the 160 days of storage (Fig. 3A), but fructoseincreased from Day 0e160 mostly because of the breakdown ofsucrose into its component sugars fructose and glucose over thelast 80 days (Fig. 3B, D). Thus the microbes preferred glucose overfructose as a carbon nutrient. Within experimental error, therewere no clear differences or trends among the various syrups withrespect to the loss of total sugars across the storage period (Fig. 3A).The rate of total sugar breakdown was higher over the first 2-daysof deterioration (av. 64.5 g L�1 Brix�1 day�1), and the rate pro-gressively decreased across the storage period with a rate of 10.6and 2.15 g L�1 Brix�1 day�1 from Days 2e40 and Days 40e160,
G. Eggleston et al. / Biomass and Bioenergy 81 (2015) 424e436 431
respectively. In general, the rate of sucrose loss was faster in clar-ified than raw syrups over the first 2 days of storage, but progres-sively became slower than for raw syrups over the 160 day storageperiod.
Low water activity (Aw) of foods is critical to the inhibition ofmicrobial growth. Foods that have higher moisture content tend tohave higher water activity though some exceptions are found [24].Regarding the microbial spoilage of food products, it is reportedthat most spoilage bacteria do not grow below Aw ¼ 0.91 and mostspoilage molds (fungi) and yeasts do not grow below Aw ¼ 0.80 and0.88, respectively [25]. Accordingly, we found that water activitieswere between 0.63 and 0.66 for 70 Brix sweet sorghum syrups,which indicates that potentially osmophilic microbes are likely togrow unless they are pre-treated before or during storage to pre-vent spoilage. Syrups of 80 Brix have a lower water activity than 70Brix syrups and, thus inhibit the growth of all but the hardyosmophilic microorganisms. Greater than 80 Brix syrups for storagewould not be practical because of their high viscosity, low flow, anddifficult handling problems. Stored, unsealed syrup will attractmoisture from the air decreasing thewater activity at the syrupeairinterface allowing the growth of osmophilic fungi. Overall, theseresults emphasize the need to focus on preventing microbialdeterioration of syrups on storage to reduce sugar losses, and 80Brix syrups stored better than 70 Brix syrups.
Changes in the pH of the raw and clarified syrups across storagein 2011 are listed in Table 3. The greatest change in pH occurredover 0e2 and 2e5 days of storage for raw and clarified syrups,
0
500
1000
1500
2000
2500
0 1 2 5 10 22 40 80 160
Suga
r Con
cent
ratio
n (g
/L o
n a
Brix
bas
is)
Days of storage
A. Total Sugars
Hybrid-A Sept rawHybrid-A Sept clarifiedM81-E Oct rawM81-E Oct clarifiedM81-E Nov rawM81-E Nov clarified
0
200
400
600
800
1000
1200
1400
1600
0 1 2 5 10 22 40 80 160
Suga
r Con
cent
ratio
n (g
/L) o
n a
Brix
bas
is
Days of storage
B. Sucrose
Hybrid-A Sept rawHybrid-A Sept clarifiedM81-E Oct rawM81-E Oct clarifiedM81-E Nov rawM81-E Nov clarified
Fig. 3. Changes in (A) total fermentable sugars (glucose þ fructose þ sucrose) measured ussweet sorghum syrups across 160 days of storage at ambient temperature (22e26 �C). ARenewables, Memphis, TN in 2011.
respectively (data not shown), and may have been linked with thedegradation of sugars in the first days of storage (Fig. 3). The pHdrop across 160 days of storage was consistently greater in theclarified than raw syrups (Table 3), which may indicate that theclarified syrups were more susceptible to microbial and chemicaldeterioration on storage. However, the sugar results in Fig. 3 did notsupport this. A better explanation is that the basic salts formed onclarification precipitated out on storage of clarified syrups,although an analysis of lactic, acetic, and formic acids and calciumsalts is still needed to confirm this. The greater decrease in pHacross 160 days of storage for clarified syrups may have aided in theprevention of growth of spoilage microbes resulting in enhance-ment of the storage shelf-life of the sorghum syrup. Kumar et al.[26] recently reported a similar beneficial decrease in pH on thestorage of pasteurized sweet sorghum juices.
3.4. Storage of sweet sorghum syrups from Louisiana (LA), 2012study
Results for storage experiments for both 70 and 80 Brix raw andclarified syrups produced from the Dale and Theis cultivars (LAstudies) are illustrated in Figs. 4 and 5 and Table 3. Similar to thesyrups from TN in 2011, all of the 70 Brix clarified syrups had fungalgrowth on their surface, especially after 80 and 160 days of storage.There was, however, a dramatic effect of the target clarification pHon the storage of clarified 70 Brix syrups (Fig. 4). The heterogeneousfeatures on the surface of the contaminated syrups at each target
0
100
200
300
400
500
600
700
0 1 2 5 10 22 40 80 160
Suga
r Con
cent
ratio
n (g
/L o
n a
Brix
bas
is)
Days of storage
C. Glucose
Hybrid-A Sept rawHybrid-A Sept clarifiedM81-E Oct rawM81-E Oct clarifiedM81-E Nov rawM81-E Nov clarified
0
100
200
300
400
500
600
700
0 1 2 5 10 22 40 80 160
Suga
r Con
cent
ratio
n (g
/L o
n a
Brix
bas
is)
Days of storage
D. Fructose
Hybrid-A Sept rawHybrid-A Sept clarifiedM81-E Oct rawM81-E Oct clarifiedM81-E Nov rawM81-E Nov clarified
ing ion chromatography, (B) Sucrose, (C) glucose, and (D) fructose in raw and clarifiedll syrups were 70 Brix and produced from sweet sorghum juice grown at Delta Bio-
Table 3Changes in storage pH of sweet sorghum syrups with different target limed (clarification) pHs, Brix, and leaf removal, fromDelta BioRenewables, TN, and USDA-ARS, Houma, LAin 2011 and 2012.
G. Eggleston et al. / Biomass and Bioenergy 81 (2015) 424e436432
pH indicated that different species of fungi colonized the syrupsurface; thus, different pHs of the stored syrups favored differentfungi. At clarification target pH 6.1, less fungi were visible (Fig. 4). In
strong contrast, none of the 80 Brix clarified syrups had visual fungigrowing on their surface at any target pH studied, which is furtherevidence that Brix and water activity are critical to syrups storage
Fig. 4. Photographs of the surface of Theis clarified 70 and 80 Brix syrups produced in Houma, LA after storage for 80 days at ambient temperature (av. 24 �C). The dark areas aresyrup. Similar results were obtained after 160 days storage.
G. Eggleston et al. / Biomass and Bioenergy 81 (2015) 424e436 433
(Fig. 4). There were some indications of microbial respiration, i.e.,bubbles, on the surfaces of the pH 6.5 and 6.8 clarified syrups, butnone at pH 6.1 (Fig. 4). Overall, these results suggest that the pH ofthe stored syrups may need to be lowered to <pH 6.1 for long-term
Fig. 5. The effect of an inexpensive, soybean oil surface layer (~15 mm) on storedM81E70 Brix syrups after 80 days storage (2012 Delta BioRenewable samples). Similar re-sults were obtained at 160 days.
storage. Wu et al. [4] reported that a low initial pH of 4.7 for a sweetsorghum juice reduced microbial growth. Also, citric acid has beenpreviously used to preserve sugarcane juice [6]. However, adjustingthe syrup pHs by adjusting the target pH of clarification will onlycause the loss of expensive fermentable sugars during the thermalclarification and subsequent evaporation processes [11]. Thus, post-evaporation adjustment of the pH of raw and clarified syrups ismore suitable and further studies are now needed on storage per-formance as well as identifying spoilage fungi and their effects onsyrup integrity throughout long-term storage.
As the target clarification pH decreased from 6.8 to 6.1, therewasa concomitant decrease in the pH drop across the 160 days ofstorage (Table 3). This strongly indicates that when less lime wasadded during clarification, less precipitation of basic, lime saltsoccurred during the storage of clarified syrups. Furthermore, the pHdrop across the total length of syrup storagewas usually lower in 70than 80 Brix syrups because the higher viscosity of the latter willhave impeded precipitation. Sugar beet syrups that are stored inbulk tanks (21e42 dam3) to extend the processing campaign, aremanufactured after carbonatation clarification of sugar beet juiceswhich also includes the minimum addition of lime salts. Duringsyrup storage it is known that the precipitation of calcium salts canoccur in the tanks [9].
3.5. Syrups from Tennessee 2012 study e effect of surfacetreatments on syrup storage
Since the syrupeair interface with the localized higher wateractivity was where fungal colonies were found, we investigated theaddition of inexpensive surface treatments to prevent microbialdeterioration on storage. Surface treatments were selected as theycould be easily (i) removed with common industrial separationsprior to fermentation and (ii) scaled to industrial production size.This is in contrast to the use of expensive broad spectrum anti-microbial additives that could later inhibit the fermentationprocess.
A thin layer of soybean oil was placed on the surface of stored 70and 80 Brix syrups to prevent undesirable microbial growth. An
G. Eggleston et al. / Biomass and Bioenergy 81 (2015) 424e436434
inexpensive vegetable oil was chosen as it is GRAS in the USAwhereas petroleum oils are not. Adding surface oil as an air-tightcoat seal on stored syrups is a practice used in the sugar beet in-dustry [10]. Oils are low density liquids that serve as a surface
Fig. 6. The changes in per cent total sugars after both 80 and 160 days of storage in (A) M8
sealant or barrier between the top of the syrup and the air abovethe syrup in the tank. As oils are low density they would not beexpected to diffuse, migrate, or sink into the stored syrup. In thisway, the oil maintains an anaerobic environment and high osmotic
1E and (B) Dale sweet sorghum syrups treated with and without surface soy bean oil.
Fig. 7. The appearance of an inexpensive, candelilla wax surface layer (~15 mm) onstored M81E 80 Brix syrups after 80 days storage (2012 Delta BioRenewable samples).Similar results were obtained at 160 days.
G. Eggleston et al. / Biomass and Bioenergy 81 (2015) 424e436 435
condition that are unfavorable to many microbes that contaminatesweet sorghum syrups. It was clearly shown in Fig. 5 that the oillayer, generally, inhibited microbial growth on both the M81E andDale syrups even when stored at 70 Brix. It was also observed thatthere was no surface microbial spoilage on syrups with oil on topafter 1 year of storage (results not shown).
Differences in the loss of total sugars across both 80 and 160days of storage for 70 and 80 Brix M81E and Dale syrups, with andwithout the addition of surface oil are illustrated in Fig. 6. As ex-pected, non-treated syrups had considerable total sugar lossesacross 80 days of storage that, generally, became worse after 160days especially for M81E syrups. Compared to the non-treatedsyrups, after 80 days storage the oil treatment prevented no ornegligible total sugar losses in raw and clarified syrups at both Brixlevels, irrespective of cultivar (Fig. 6). Some of these syrups had aportion of their sucrose break down into glucose and fructose, butoverall no total sugars were lost. Furthermore, some M81E syrupstreated with surface oil did deteriorate from 80 to 160 days, butDale syrups did not show any significant deterioration (Fig. 6).
The surface oil layer also reduced the pH drop across 160 days ofstorage of raw syrup compared to no oil (Table 3), but there was nosignificant correlation of the pH drop with the degradation ofsugars. In contrast, there were often negligible differences in pHdrops on storage of clarified syrups treated with or without oil,because the pH drop in clarified syrups is governed by the pre-cipitation of basic salts. Similar to previous results in this study, pHdrops across 160 days of storage were lower in stored 80 than 70Brix syrups, which is further evidence that less deteriorationoccurred in 80 than 70 Brix syrups.
Overall, these results suggest that 70 Brix raw or clarified syrupswith a thin surface of GRAS vegetable oil could be stored long-term,mitigating the energy costs associated with creating high Brixsyrups as well as unwanted viscosity problems. One disadvantageof the oil layer is its removal, but syrup could be removed from thetank below the oil level. Furthermore, industrial oil skimmers arereadily available and well-known for their high performance,sturdy construction, and long service life. They are widely used inmany industries including sewerage and industrial water in-dustries. Future storage studies are now needed, including the useof other inexpensive vegetable oils with varying properties onlower Brix syrups.
The effects of a layer of an inexpensive wax on the surface ofstored 80 Brix syrups were also investigated, as wax may be easierto remove than oil. Additionally, waxes have the added benefit inthat they are recyclable and can be imbued with anti-microbialcompounds [27]. In this study, edible, candelilla wax was usedwhich has inherent anti-microbial properties and is FDA-GRASapproved. Candelilla wax is derived from the candelilla plant(Euphoria antisyphilitica Zuce) [28]. The use of candelilla wax as acoating on stored apples to inhibit fungal growth and reducing lossof water vapor was demonstrated by Ochoa et al. [28]. Candelillawax was able to stop the spoilage of 80 Brix syrups (Fig. 7) andserved dual purposes as (i) a surface sealant and (ii) a syrup pre-servative. Typically, there were no significant differences in the pHdrop during storage for raw syrups with a wax layer thanwithout awax layer. There was evidence that for clarified syrups the pH dropwas greater than in raw syrups which is further evidence of theprecipitation of basic salts. Moreover, for clarified syrups the waxlayer may have slightly improved the pH drop during storage(Table 3). One slight disadvantage of using candellila wax surfacetreatment is the energy required to melt the wax at 68.5 to 72.5 �C[29]. Another disadvantage of using wax is the possible breaking ofthe wax layer when syrup is removed from below it. Future storagestudies involving lower Brix syrups and other inexpensive waxesare now warranted.
4. Conclusions
Leaving leaves on the sweet sorghum stalk actually aided theclarification of juice, particularly reducing the settling time, andmay enhance sugar yields. Overall, the processing differences withleaves removed compared to no removal, were not great enough toconsider removing more leaves. At the pilot plant scale, ~92e98%turbidity removal across clarification of sweet sorghum juice(80 �C; target limed juice pH 6.3; 5 ppm polyanionic flocculant) wasobtained after only 30e50 min of clarification and was highlydependent on cultivar. Clarification of the juices reduced the loss offermentable sugars during the subsequent evaporation stage, andhad the added advantage of stabilization and syrup storage.
The surface of the syrup was the most susceptible to fungaldeterioration on storage due to evaporation and condensation.Sweet sorghum syrups at 80 Brix had lower water activity than 70Brix syrups and were less susceptible to microbial spoilage duringstorage. At the industrial site, however, 80 Brix syrups will be moredifficult to handle and transport due to slower flow and higherviscosity characteristics. Target clarification pH had a dramatic ef-fect on the storage of clarified 70 Brix syrups with more acidic pHsreducing fungal deterioration and further studies are now war-ranted on the post-evaporation pH adjustment of raw and clarifiedsyrups to <6.1 for long-term storage. Inexpensive surface treat-ments of a thin layer of either soybean oil or candellila wax whichare both GRAS, showed strong potential to prevent microbialcontamination of syrups and now warrant further investigationwith 65e70 Brix syrups at the large, industrial scale. Large scalestudies will require optimizing (i) the filling of the bulk tanks, (ii)the tank design, and (iii) finding the best management practices formonitoring the stored syrups.
G. Eggleston et al. / Biomass and Bioenergy 81 (2015) 424e436436
Acknowledgments
The authors are grateful to the United Sorghum Checkoff Pro-gram (USCP RN001-14) for funding this research. Mention of tradenames or commercial products in this article is solely for the pur-pose of providing specific information and does not imply recom-mendation or endorsement by the U.S. Department of Agriculture.USDA is an equal opportunity provider and employer.
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