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Research Project: Developing Technologies that Enable Growth and Profitability in the Commercial Conversion of Sugarcane, Sweet Sorghum, and Energy Beets into Sugar, Advanced Biofuels, and Bioproducts

Location: Commodity Utilization Research

2017 Annual Report

The overall objective of this project is to enhance the value of sugarcane, sweet sorghum, and energy beets, and their major commercial products sugar, biofuel and bioproducts, by improving postharvest quality and processing. Specific objectives are: 1. Develop commercially-viable technologies that reduce or eliminate undesirable effects of starch and color on sugar processing/refining efficiency and end-product quality. 2. Develop commercially-viable technologies that reduce or eliminate undesirable effects of high viscosity on sugar processing/refining efficiency and end-product quality. 3. Develop commercially-viable technologies to increase the stability and lengthen storage of sugar feedstocks for the manufacture of sugars, advanced biofuels, and bioproducts. 4. Develop commercially-viable technologies for the biorefining of sugar crop feedstocks into advanced biofuels and bioproducts. 5. Identify and characterize field sugar crop quality traits that affect sugar crop refining/biorefining efficiency and end-product quality, and collaborate with plant breeders in the development of new cultivars/hybrids to optimize desirable quality traits. 6. Develop, in collaboration with commercial partners, technologies to improve the efficiency and profitability of U.S. sugar manufacturing and enable the commercial production of marketable products from residues (e.g., bagasse, trash) and by-product streams (e.g., low purity juices) associated with postharvest sugar crop processing. Please see Project Plan for all listed Sub-objectives.

There are currently two major trends in the U.S. with respect to sugar crops: (1) the manufacture of higher quality raw sugar for supply to sugar refineries, and (2) the production of biofuels and bioproducts at new, flexible biorefineries. In recent years, mostly because of the increased harvesting of green sugarcane with leaves and tops, higher concentrations of starches and color have tended to occur. Some U.S. sugar refiners have placed a penalty for high starch concentrations in raw sugar. The occurrence of larger concentrations of insoluble starch in downstream factory products have exacerbated viscosity problems and reduced the efficiency of amylase enzymes to control starch. In close collaboration with industrial partners ARS scientists will develop new enzyme systems and other commercially viable technologies to control starch, viscosity, and color in factory and refinery streams, while also developing a method for measuring both insoluble and soluble starch in sugar products at the factory and refinery. Stable, storable, easily transportable, and available year-round supplies of sugar crop feedstocks, including sweet sorghum and energy beets, are needed for the conversion of sugars into substitute biofuels and bioproducts normally manufactured from fossil products. In close collaboration with industrial partners, ARS scientists will develop commercially-viable technologies for the extraction, stabilization, concentration, and fermentation of juices and syrups from sweet sorghum and energy beet feedstocks that will enable the deployment, growth, and profitability of new commercial biorefineries. Commercially-viable technologies will also be developed that are crucial to mitigate cultivar, seasonal, and environmental quality variations on feedstock performance.

Progress Report
Conversion of Sugar Crop Solutions to Biofuel and Bioproducts (Objective 4). The biofuels and bioproducts ethanol, butanol, succinic acid (precursor to 1,4-butanediol), and acetoin were tested with simulated sugar solutions and diluted sugar crop syrups in bench-scale reactors. All products could be produced from the simulated solutions. To aid in the work, bench-scale equipment was modified so that multiple fermentation experiments could be conducted under controlled conditions. Aconitic acid (a potential fermentation inhibitor) was the only major organic acid found in the commercial sugar solutions and sweet sorghum juices. Under normal fermentation conditions, aconitic acid does not impact fermentation; however, if the conditions are very acidic (like the unique operation conditions in Brazilian sugarcane bioethanol plants) it will have a negative impact. A portion of the ethanol research was presented at the Symposium on Biotechnology for Fuels and Chemicals Meeting on Advances in Sugar Crop Processing and Conversion, and at the Annual Meeting of the Society of Industrial Microbiology and Biotechnology. Conversion of Sugar Beet Juice Sugars to Acetoin (Objective 4). Acetoin is a compound that contributes to the flavor of butter, and is used as a food additive to impart that quality to baked goods and other foods. We demonstrated that two Bacillus strains of bacteria could convert glucose in beet thick juice (syrup) to acetoin under laboratory-scale fermentation conditions. Beet juice was mixed with decreasing concentrations of glucose, and data showed that beet juice alone was enough to support conversion of beet sugar to acetoin. These results confirmed that the beneficial product acetoin can be cost-effectively produced from beet juice with little to no supplementation with added sugar. Future work will include scaling up this technology. Identification of New Uses for Sugarcane Biochar (Objective 3). Vermiculture (composting using worms) is sometimes used to improve soil quality since worm castings (organic fertilizer) are known to enhance soil quality and structure through both beneficial microbes and soil nutrients. To commercialize worm castings as a soil amendment, it is necessary for these characteristics to be stabilized through storage. We demonstrated the storage stability and more effective dispersal of the worm castings onto soils, by producing blends with sugarcane biochar at different ratios. While microbial counts for worm castings significantly decreased during storage, additions of up to 25% of biochar doubled microbe counts during a 3 month storage study. In a subsequent study it was determined that blends containing up to 75% biochar did not significantly inhibit microbial stability. The addition of biochar also increased the carbon content of the blends and improved soil structure. Sugarcane biochar produced from bagasse (fibrous by-product) and leafy trash at a Louisiana sugarcane factory was also applied to fields growing both plant (first year) cane and first ratoon (second year) cane at three commercial farms. Biochar was applied in three different forms: powder with molasses, powder without molasses, and pellets with molasses. Sugarcane was hand-harvested and juice extracted. Data is currently being analyzed and the fields will be monitored for an additional year. It is expected that the addition of biochar to the field will enhance soil quality, improve sugarcane growth and/or sugar yield. Biochar was also produced from sugarcane bagasse and leafy residues in a pilot plant and applied it to a grower’s field. Fly ash (residue from factory boilers) was also added as a treatment to determine its contribution to the yield of the sugarcane crop compared to the biochar. Mixtures of fly ash and biochar were applied at two levels and compared to no treatment. Plant cane (first year growth), first ratoon (second year growth), and second ratoon (third year growth) crops were harvested in a three year field study and juices was extracted with roller milling. Analysis included stalk length and weight, and juice was analyzed for Brix (% soluble solids), purity, and fiber content. Statistical analysis of all data has been completed. Although there were no significant differences between treatments, biochar improved the ratooning (re-growth) ability of sugarcane over the three year period. Additionally fly ash addition to soil did not have a deleterious effect in sugarcane or sugar yield. One additional year will be harvested and data collected for this study. Final progress report for Agreement 58-6054-6-0021 (Objective 1). The application of chemical ripeners is an important component of sugarcane cultivation management in the United States to increase sucrose concentrations. Little information was available on the effects of ripener on starch and color quality parameters critical to both factory and refinery processing. A large, two-year field study was conducted on the effect of two chemical ripeners with differing modes of action on sugarcane: Polado (glyphosate chemical) that suppresses the formation of new tissue at the top of the stalk, and Moddus (trinexapac-ethyl chemical) that interferes with stalk elongation. The ripeners were applied to nine commercial sugarcane varieties cultivated in South Louisiana, USA, and hand-harvested 4 to 7 weeks after ripener application. Results from the first year of the study showed that adding either ripener increased total starch in stalks by 20.7%, although this was not statistically significant because of a very strong varietal effect. Field variability in the starch values was much higher than variability for Brix (percent soluble solids), pH, and color values, even in the untreated juices. The ripeners had less effect on pH and color than starch. No significant differences occurred with ripener treatment for color measured at pH 4.0, 7.0, 8.5, and 9.0, although color variability increased with ripener application. Analysis of the second year of study is currently being completed to verify the first year trends. Progress report for Agreement 6054-41000-110-25N in collaboration with Louisiana State University (Objective 6). Activities included the initial stage planning of a full day workshop to be held at ARS, New Orleans, Louisiana, as part of the Water Environment Technical Annual Conference and Exposition (WEFTEC) in September 2018. A pre-proposal was submitted in July 2017. This workshop will bring together various experts in the field of thermochemical biomass conversion into value-added products and energy. Progress report for Agreement 58-6435-4-005 (Objective 1). In collaboration with the National Agriculture and Food Research Organization, the Ministry of Agriculture, Forestry, and Fisheries, Japan, specialized methods based on chemical oxidation-reduction probes and nuclear magnetic resonance spectroscopic analyses. The methods were then successfully used to identify the chemical functionalities responsible for the antioxidant capacity of sugarcane-derived value-added products, and different forms of phosphorus nutrients in agricultural wastes. Final progress report for Agreement 58-6054-5-0021 (Objective 1). Near Infrared Spectroscopy (NIRs) that rapidly measures the amount of various quality parameters in sugarcane juice and biomass has potential to benefit the U.S. sugar industry. In a two year study, together with an industrial collaborator, we took core samples from three Louisiana factories each week across the 3-month processing season. The samples were transported to the NIR pilot plant instrument and analyzed and compared to conventional juice analyses at the factory. All the pressed-out juices were also analyzed for color at pH 4, 7, 8.5, and 9, conductivity ash, and mannitol. The levels of leafy trash in samples were also shown to be detected by NIR which means there is high potential for NIR to be used as an automatic cane payment system in Louisiana. Final report for Agreement 58-6054-5-0022 (Objective 1). We completed the development of a new method to rapidly, precisely, and accurately measure total, soluble, and insoluble starch at the factory and refinery. The method is based on chemical assisted microwaving and the estimated cost is only 4 cents per analysis. The method has already been submitted as a new method to the International Commission for Uniform Methods in Sugar Analysis and is being transferred to factories. Final progress report for Agreement 58-6054-6-019 (Objective 6). In collaboration with West Virginia State University, biochars were developed from sugar crop residues to make sorbent materials, and both the feedstock and pyrolysis conditions were evaluated for their ability to remove selected heavy metals and oxyanions from water. Findings were presented at the Association of 1890 Research Directors 18th Research Symposium, 2017, in Atlanta, Georgia, and at the 2017 American Society of Sugar Cane Technologists meeting in New Orleans, Louisiana, and a manuscript is being written. Final progress report for Agreement 6054-41000-110-01O (Objective 4). In collaboration with Oak Ridge Institute for Science and Education, a special enzyme system was developed to convert starch in processing products, by-products, and waste materials from sweet sorghum syrup manufacturing. The developed enzyme system was able to convert all the starch into sugars. Application of the enzyme method followed by fermentation of the syrup to ethanol by yeast, increased the ethanol yield by up to 2.5 fold.

1. Established the continuous use of a High Performance Liquid Chromatography (HPLC) system at a sugarcane factory to accurately and rapidly measure sugars. On request from industry, ARS researchers in New Orleans, Louisiana, in collaboration with a Louisiana sugarcane factory and Louisiana State University, established the first HPLC at a Louisiana sugarcane factory. A HPLC sugar method was developed and established to measure mannitol, glucose, fructose, and sucrose in a run time of 10 minutes at the factory. For the best accuracy, separate and higher dilutions are needed to quantitate sucrose due to its considerably higher concentration in sugar products (except molasses). Training was formalized and conducted because it was essential for operations and analyses and this included basic sugar chemistry, major sugarcane deterioration reactions, and color formation in the factory, since an analytical technique is only useful if the results are interpreted properly. A simplified chart was created and provided to the factory staff to help interpret the results. The HPLC system allowed the factory to: (i) monitor sucrose losses in “real time”, (ii) rapidly identify sugarcane deterioration via mannitol (sugar alcohol) measurement, (iii) rapidly monitor loss of sucrose in molasses and dextranase (enzyme that breaks down unwanted dextran polymer) applications, and (iv) explain difficult samples more easily. In the 2016 processing season, the use of HPLC allowed the detection of excess sucrose losses of approximately 0.49%, in one of three juice clarification tanks at the factory, which is conservatively equivalent to $460,000 loss in sucrose yields per year. This caused the factory staff to retrofit the tank to reduce heat spots and sucrose losses, and this alone has paid for the cost of the HPLC system.

2. Ensiling sweet sorghum bagasse. As the U.S. production of sweet sorghum increases, there are associated increases in the accumulation of low value bagasse which is a fibrous by-product. Processers need to quickly stabilize the bagasse after it is produced so it can be converted into useful bioproducts after the harvest season. ARS researchers in New Orleans, Louisiana, used ensile (preservation of biomass in a silo) technology to store sweet sorghum bagasse. This was successful and resulted in anaerobic conditions in which the bagasse can be stably stored for subsequent use as animal bedding. Utilizing inexpensive, commercially available compacting equipment, an anaerobic environment was successfully created and maintained that was sufficient enough to inhibit microbial degradation of the stored bagasse. Key factors identified were the compaction rate and the thickness of the packaging material surrounding the bales. Based on the processors’ standard milling practices, bagasse from both single and double pass millings were ensiled. It was determined that although single pass bagasse did stabilize, double pass bagasse was ensiled more efficiently because it was finer and easier to compact. The collaborating sorghum producer is now using the demonstrated conditions to scale-up to commercial scale.

3. Identification of quality and processing traits in sweet sorghum to aid breeders and industrial processors. Since 2013, insects such as sugarcane aphids have infested sweet sorghum in the Midwest and Southeast USA, considerably reducing crop yields. ARS researchers in New Orleans, Louisiana, and Tifton, Georgia, discovered that the chemical compounds trans-aconitic and oxalic acids were found to be quality traits of sweet sorghum which indicate pest resistance to sugarcane aphids. Chemical fingerprints for trans-aconitic acid and oxalic acid can be used to rapidly screen for resistant lines to develop new sweet sorghum cultivars, and aphid resistant lines have already been identified. Higher yields of trans-aconitic and oxalic acids and other secondary products (dissolved salts, organic acids, phenolics, and amino acids) were accompanied by lower yields of primary products (fermentable sugars) in sweet sorghum stalk juice. The planting date was also important. These findings have provided a recommendation for Southern Coastal Plane growers to employ later planting date (from April to June) to maximize the stem sugar yields, depending on the degree of interactions between the cultivar and the planting date.

4. Evaluation of commercial biocides in sugarcane juice to determination of economic feasibility. The microbial contamination of extracted sugarcane juice is a serious problem in raw sugar manufacture because it causes expensive sugar losses and the formation of degradation products that interfere with processing. In 2016, U.S. sugarcane factories requested information on biocides to control bacterial growth during milling. ARS researchers in New Orleans, Louisiana, in collaboration with a sugarcane factory and a processing aid company, evaluated potassium permanganate as a biocide. Potassium permanganate was shown to reduce the growth of both gram negative and positive bacteria in sugarcane juice and bagasse. The combination of hydrogen peroxide and sodium hypochlorite chemicals also reduced bacterial growth and additionally decolorized juice. An economic assessment of these findings from the factory has provided a conservative estimate that if potassium permanganate is added on a 24 hour basis at a sugarcane factory it could save $280,000 in sucrose losses alone per year. Further studies are needed before implementation at the factory.

5. Causation of floc formation in alcohol beverages sweetened with refined cane sugars. Alcohol beverages are high value-added products with large domestic and international markets. Consumers expect the beverage to be clear and will most likely be deterred from purchasing the beverage if flocs (suspended large particles) are present. Unfortunately, with the declining use of high fructose corn syrup as a beverage sweetener in the USA, the sporadic appearance of floc from refined cane sugars has been increasing and it remains a technical problem that is not easily managed. ARS researchers in New Orleans, Louisiana, were approached by a large American alcohol manufacturer to improve understanding of the root cause or causes of floc formation. Insoluble and soluble starch, fat, inorganic ash, oligosaccharides (short chain sugars), percent soluble solids, and pH were shown not to be involved in the prevailing floc formation mechanism. There were strong relationships between floc formation and protein content of the sugar as well as the sugar’s color indicator value which is an indirect measure of polyphenolic and flavonoid colorants. The ethanol in the beverages were shown to induce the denaturation of the protein and create more hydrophobic polyphenol binding sites and, therefore, more floc. Polyphenol-protein flocs are known to occur in ciders, wines, and beers. A tentative mechanism for floc formation was advanced by molecular probing with a floc active protein and polyphenol, as well as polar, non-polar, and ionic solvents. By knowing the cause of floc formation solutions, such as activated carbon, have now been put forward to removing these components at the sugar refinery or at the distillery. Cane Land Distilling Company in Baton Rouge, Louisiana, is already using these research findings.

6. Development of an industrial method to measure insoluble and soluble starch in sugar products at a sugarcane factory or refinery. In recent years, starch impurity concentrations in sugarcane raw sugar have been increasing in the United States. ARS researchers in New Orleans, Louisiana, showed that existing starch methods used in the international sugar industry do not accurately measure total starch because they cannot efficiently solubilize the insoluble starch well and are also limited by the color of the factory product. At the request of industry, a rapid, precise, and accurate industrial method was developed, based on chemical assisted microwaving, to measure total, soluble, and insoluble starch at the factory and refinery. The industrial method compared favorably (less than 6.5% difference in accuracy and precision) with the USDA Starch Research Method but is much less expensive because the relatively expensive probe sonicator in the USDA Starch Research Method was replaced with less expensive and readily available chemicals. The new method costs only 4 cents per analysis. The method has already been submitted as a new method to the International Commission for Uniform Methods in Sugar Analysis.

7. Development of a novel starch breakdown enzyme system to increase fermentation yields from by-products obtained during sweet sorghum syrup manufacture. In collaboration with an industrial collaborator, ARS researchers in New Orleans, Louisiana, identified seeds, bagasse (fibrous product from roller milling), juice sediment and clarification mud, both by-products of sweet sorghum large-scale processing, as rich in starch. Thus these industrial by-products could be used as untapped sources of fermentable sugar. A hydrolysis and saccharification (starch breakdown) system consisting of five enzymes was developed specifically for the sweet sorghum industry and found to increase fermentable sugars by 50-75% and double ethanol yields using the industrial by-products. It was conservatively estimated that less than $20 worth of enzymes would be needed per ton of starchy by-products. It was also found that chemical or microbial impurities usually concentrated in these by-products did not negatively affect laboratory hydrolysis, saccharification, and fermentation experiments. These findings have also been successfully applied to the primary industrial products of sweet sorghum juices and syrups.

8. Development of technologies to control color in product streams at the sugarcane factory and refinery. The removal of naturally-derived or process-derived sugarcane colorants from raw sugar, by separation methods or chemical breakdown methods, is the primary goal of sugar refiners. ARS researchers in New Orleans, Louisiana, developed new advanced oxidation processes (AOPs) to enhance product decolorization at sugarcane factories where there are less stringent constraints to the addition of processing aids compared to the refinery. Four different AOPs (permanganate, hydrogen peroxide, persulfate, and peroxymonosulfate chemicals) were screened under different processing conditions. The permanganate exhibited an order of magnitude greater decolorization than the other AOPs. In addition to chemically breaking down the cane-derived colorants by forming reactive free radicals, permanganate enhanced the juice clarification processes by forming colloidal particles. The findings could be used by sugarcane factories but further factory studies are now needed.

9. Starch formation and interference with carbonatation clarification at a sugar refinery. Raw sugars with >250 parts per million (ppm) total starch (long chain polysaccharide) cause problems during clarification (using carbonate) at the refinery. Using raw sugars with a large range in quality, ARS researchers in New Orleans, Louisiana, demonstrated that existing analytical methods used to predict refining performance do not accurately represent the processing challenges from high-starch concentrations. By conducting a large, three-part study, it was determined that: (i) insoluble starch (starch form before it is swollen) is most deleterious to press filtration after the clarification process, and (ii) soluble starch binds calcium ions and limits the formation of calcium carbonate mud, thus producing small calcium carbonate crystals which make filtration worse. Raw sugars containing 35-65% insoluble starch were found to be more deleterious to carbonatation clarification and press filtration, although soluble starch disrupted the carbonatation chemical reactions. These findings support the need for new refinery methods to accurately depict both soluble and insoluble starch’s behavior during carbonatation clarification and press filtration as well as the need to measure total, soluble, and insoluble starch in raw sugars and refinery products.

10. Identification of fermentation inhibitors. Aconitic acid, naturally present in sweet sorghum juice and syrup, has been mentioned as a fermentation inhibitor that slows down biofuel production rates but very little proof existed. ARS researchers in New Orleans, Louisiana, with a commercial partner proved that juice clarification (chemical treatment of juice to remove a variety of turbid compounds) did not remove aconitic acid. The ARS researchers also confirmed that fermentation inhibition existed and that it was linked to one of four chemical versions of aconitic acid that only occurs in acid environments. This explains why some researchers have found that aconitic acid was an inhibitor to fermentation while other researchers did not. This is important because it provides the emerging sweet sorghum juice and syrup fermentation industry with one solution to the problem of slow fermentation rates, which is to carry out the fermentation in less acid environments.

Review Publications
Wright, M.S. 2017. Effect of a biocide treatment on microbes in sweet sorghum juice. African Journal of Agricultural Research. 12(13):1074-1078.
Klasson, K.T. 2017. Impact of potential fermentation inhibitors present in sweet sorghum sugar solutions. Sugar Tech. 19(1):95-101.
Uchimiya, M., Hiradate, S., Chou, C.C. 2016. Polyphenolic reductants in cane sugar. Austin Food Sciences. 1(5):1022.
Klasson, K.T. 2017. Biochar characterization and a method for estimating biochar quality from proximate analysis results. Biomass and Bioenergy. 96:50-58.
Kim, K.-H., Kumar, P., Szulejko, J.E., Adelodun, A.A., Junaid, M.F., Uchimiya, M.M., Chambers, S. 2017. Toward a better understanding of the impact of mass transit air pollutants on human health. Chemosphere. 174:268-279.
Muley, P.D., Henkel, C.E., Aguilar, G., Klasson, K.T., Boldor, D. 2016. Ex situ themo-catalytic upgrading of biomass pyrolysis vapors using a traveling wave microwave reactor. Applied Energy. 183:995-1004.
Cole, M.R., Eggleston, G., Petrie, E., Uchimiya, S.M., Dalley, C. 2017. Cultivar and maturity effects on the quality attributes and ethanol potential of sweet sorghum. Biomass and Bioenergy. 96:183-192.
Cole, M., Eggleston, G., Triplett, A. 2017. Analytical evaluation of current starch methods used in the international sugar industry: Part I. Food Chemistry. 228:226-235.
LeBlanc, J., Uchimiya, M., Ramakrishnan, G., Castaldi, M.J., Orlov, A. 2016. Across-phase biomass pyrolysis stoichiometry, energy balance, and product formation kinetics. Energy and Fuels. 30:6537-6546.
Guo, M., He, Z., Uchimiya, S.M. 2016. Introduction to biochar as an agricultural and environmental amendment. In: Guo, M., He, Z., Uchimiya, S.M., editors. Agricultural and Environmental Applications of Biochar: Advances and Barriers. Soil Science Society of America Special Publication 63. Madison, WI:Soil Science Society of America. pp. 1-14.
Wright, M.S., Lima, I.M., Bigner, R.L. 2016. Microbial and physicochemical properties of sugarcane bagasse for potential conversion to value-added products. International Sugar Journal. 118:424-433.
Guo, M., He, Z., Uchimiya, S.M. 2016. Preface for "Agricultural and environmental applications of biochar: Advances and barriers." In: Guo, M., He, H., Uchimiya, S.M., editors. SSSA Special Publication 63. Madison, WI:Soil Science Society of America, Inc. p. v-viii.
Boone, S., Klasson, K.T., St Cyr, E., Montes, B., Pontiff, K., Legendre, D., Wright, M. 2017. Limiting sucrose loss in Louisiana raw sugar factories: Are biocides necessary? International Sugar Journal. 119(1420):288-293.
Bhattacharya, S.S., Kim, K.H., Das, S., Uchimiya, M., Jeon, B.H., Kwon, E., Szulejko, J.E. 2016. A review on the role of organic inputs in maintaining the soil carbon pool of the terrestrial ecosystem. Journal of Environmental Management. 167:214-227.
Eggleston, G., Wartelle, L., St Cyr, E. 2016. Detecting adulterated commercial sweet sorghum syrups with ion chromatography oligosaccharide fingerprint profiles. Separations. 3(20):1-16.
Uchimiya, M., Wang, M.L. 2016. Roles of conjugated double bonds in electron-donating capacity of sorghum grains. African Journal of Agricultural Research. 11(24):2146-2156.
Cole, M.R., Eggleston, G., Rathke, T., Naiki, J., Triplett, A., St Cyr, E. 2016. How the physical forms of starch affect filterability at a carbonatation refinery. Part II: simulated carbonatation filtration. International Sugar Journal. 118:650-658.
Guo, M., Uchimiya, S.M., He, Z. 2016. Agricultural and environmental applications of biochar: Advances and barriers. In: Guo, M., Uchimiya, S.M., He, Z., editors. Agricultural and Environmental Applications of Biochar: Advances and Barriers. SSSA Special Publication 63. Madison, WI:Soil Science Society of America. pp. 495-504.