Sweet Sorghum Improvement and Production in Brazil Consultation on Pro-poor Sweet Sorghum Development for Bio-ethanol - IFAD November 8-9, 2007 Robert E. Schaffert Embrapa Milho e Sorgo schaffer@cnpms.embrapa.br www.cnpms.embrapa.br

In the late 1970´s Brazil initiated a bio-energy program (Pro-Alcohol) anticipating an energy crises caused be a shortage of petroleum to meet Brazil's fuel needs. There was a strong incentive to develop technology for micro-distilleries (100L hr-1) and mini-distilleries (1000L hr-1) Embrapa´s sweet sorghum program was developed to provide raw material for these distilleries. Pilot Projects were successfully developed in the mid-1980’s at Sete Lagoas, Brasilia, and Pelotas to process sweet sorghum in micro-distilleries.

(Embrapa Maize and Sorghum) The potential uses of sweet sorghum for food, fiber, fertilizer, ethanol, and methane gas production (Embrapa Maize and Sorghum) xxxx Silage

Advantages of Using Sweet Sorghum vs. Sugarcane Sweet sorghum may be harvested 3 – 4 months after planting Sweet sorghum production can be completely mechanized The sweet sorghum crop can be established from seed The grain from sweet sorghum can be used as food, feed or fuel The bagasse from sweet sorghum has a higher biological value than the bagasse from sugarcane when used as a forage for animals Sweet sorghum is more water use efficient

Integrated Rural Energy System Developed at Sweet Sorghum Sugarcane Micro- distillary Other uses Integrated Rural Energy System Developed at Embrapa Maize and Sorghum 1980 Integrated rural energy system developed at Embrapa Maize and Sorghum 1980 xxxxxxxx

Development of Sweet Sorghum Cultivars Breeding priorities depend upon how the product will be used Panicles removed for food or feed Small vs. large panicles Juice is extracted from stalks and leaves Not feasible to separate leaves and stalks Juice fermented in micro- and mini-distilleries Juice extraction less efficient in smaller distilleries Bagasse used for forage or fuel Starch present in sorghum juice and stalk Biological value of sweet sorghum bagasse is greater for sweet sorghum than for sugarcane Tillering vs. Non-tillering to control stalk diameter

A Simple Flow Diagram of a Roller Mill Micro-distillery; Used in Establishing Breeding Priorities at Embrapa Maize and Sorghum Xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx X x A - roller mill, B - boiler, C - yeast treatment and distribution tank, D - juice distribution tanks, E - fermentation tanks, F – beer holding tank, and G - distillation column (Embrapa Maize and Sorghum)

Industrial Planning J F M A M J J A S O N D Sweet Sorghum Planting Harvesting Sugarcane Planting and harvesting periods for sweet sorghum and sugarcane in Brazil. (Embrapa Maize and Sorghum) J F M A M J J A S O N D Ratoon Harvest Irrigation Required

Yield and Quality Goals must be Established Minimum Total Sugar Extraction – 80 kg 1t-1 biomass Considering 60-65% Extraction Efficiency Minimum Biomass Yield - 40 tha-1 Minimum Alcohol Yield – 40L t-1 biomass Considering: 60 -65% sugar extraction efficiency 90% Fermentation Efficiency 90% Distillation Efficiency or 81% Industrial Efficiency Minimum Total Sugar Content in Juice – 12.5% Establish minimum parameters for determining Period of Industrial Utilization (PIU - 80 kg 1t-1 biomass) Minimum PIU – 30 days

Total Biomass Production of Two New Sweet Sorghum Varieties Developed at Embrapa for Micro-distilleries *

Sweet Sorghum Nomenclature Average values of brix, total invert sugars, juice extraction, and fiber of selected sweet sorghum varieties grown at Araras, São Paulo, Brazil in 1981. Variety Brix Total invert sugars (% juice) Extraction (% sorghum stalks) Fiber (% sorghum stalk) Brandes 16.7 14.7 63.2 12.5 Honey 13.0 11.1 73.0 11.6 Sart 12.4 69.8 14.8 Rio 16.4 14.2 57.6 16.1 MN 1500 11.4 61.1 18.5 MN 1048 14.1 13.4 56.5 22.0 MN 1030 15.2 10.0 47.6 25.8 MN 4008 10.6 10.5 74.9 12.0 Williams 13.5 70.5 10.1 MN 4080 ----- 45.7 26.6 Wray 19.3 16.8 67.5 Theis 71.5 Redlan 7.4 67.0 13.6 Tx623 10.8 7.0 64.0 Source: Schaffert and Borgonovi (1980)   Sweet sorghum variety names that begin with the letter or sound or R are high sucrose types and variety names that begin with other letters are low sucrose types. Redlan and Tx623 are non-sweet juicy stem female lines.

Xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx x RIO PIU – 32 days Xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx x XXX The interaction of refractometry Brix and percent total reduced sugars in the juice and percent fiber, percent juice extraction, and percent sugar extraction of sorghum stalks during the maturity phase for the varieties Rio and Wray grown in Brazil, (Embrapa Maize and Sorghum) xxxxxxxxxxxxxxxxxxx WRAY PIU – 58days Xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx Zzzz

xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx Sugar extracted ( Kg 100 Kg biomass) -1 Percent water in the biomass Percent fiber of fresh biomass xxx xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx Total Sugars (%) Xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxx Brix, Total Sugars, Fiber and Percent Water of four sweet sorghum cultivars at Embrapa Maize and Sorghum, Sete Lagoas, Brazil, 1986/87. BR 501 = Brandes, BR 505 = Wray, BR506 and BR507 are new derived varieties Total sugars (% juice) Brix (% juice)) BR 505

Period for Industrial Utilization Wray 9% Total sugars Wray 8% Total sugars xxxx Wray PUI 32 days Wray PUE 51 days xxxxxxxxxx

PUI 28 days The interaction of refractometry Brix and percent total invert sugars in the juice and percent fiber, percent juice extraction, and percent sugar extraction of sorghum stalks during the maturity phase for the variety Rio grown in Brazil (Embrapa Maize and sorghum) .

Brix, total sugars (%), fiber (%), and juice extraction (%) Sugar extraction (%) PUI – 35 days The interaction of refractometry Brix and percent total invert sugars in the juice and percent fiber, percent juice extraction, and percent sugar extraction of stalks during the maturity phase for the variety Brandes grown in Brazil (Embrapa Maize and Sorghum).

Brix, total sugars (%), fiber (%), and juice extraction (%) CMSXS 623 (Brandes Derivative) Brix, total sugars (%), fiber (%), and juice extraction (%) Sugar extraction (%) PUI 37 days The interaction of refractometry Brix and percent total invert sugars in the juice and percent fiber, percent juice extraction, and percent sugar extraction of stalks during the maturity phase for the variety Brandes grown in Brazil (Embrapa Maize and Sorghum). Days after planting The interaction of refractometry Brix and percent total invert sugars in the juice and percent fiber, percent juice extraction, and percent sugar extraction of sorghum stalks during the maturity phase for the variety CMSXS 623 grown in Brazil (Embrapa Maize and sorghum).

PUI 54 days The interaction of refractometry Brix and percent total invert sugars in the juice and percent fiber, percent juice extraction, and percent sugar extraction of stalks during the maturity phase for the variety Brandes grown in Brazil (Embrapa Maize and Sorghum). The interaction of refractometry Brix and percent total invert sugars in the juice and percent fiber, percent juice extraction, and percent sugar extraction of sorghum stalks during the maturity phase for the variety Wray grown in Brazil (Embrapa Maize and sorghum) .

Biomass and Alcohol Production of two new Cultivars, BR506 and BR507, Compared to Brandes and Wray (t ha-1) Extracted Sugar Yield (kg 100 kg-1) (t ha-1) Alcohol Yield Ideal (L ha-1) 81% Efficient Relative (% Wray) Brandes Wray BR506 BR507 47.4 44.6 48.8 52.0 7.2 10.9 9.7 9.6 3.4 4.6 4.7 5.0 2208 2946 3062 3231 1788 2386 2481 2617 75 100 104 110 Embrapa Maize and Sorghum 1986/87

Current Plant Breeding Strategies and Priorities Develop sweet sorghum 3 dwarf A and B lines (female) to be used in hybrid development and production - Identify molecular markers for juicy stem and sweet juice - Incorporate genes for multiple stress (biotic and abiotic) resistance Evaluate existing new sweet sorghum cultivars (approx. 50) and develop new sweet sorghum R lines (male) to be used in hybrid development and production - Incorporate genes for multiple stress (biotic and abiotic) resistance Identify molecular markers for tillering - Non-tillering desired to be able to control stalk diameter with plant population (better extraction with large stem diameter) Develop transgenic sweet sorghum with sucrose isomerase gene (SI), a gene that regulates the transformation of sucrose to isomaltulose and thus can increase the sink capacity of sugar storage (Wu and Birch, Plant Biotechnology Journal (2007) 5 (pp109-117)

Plant Breeding Strategies and Priorities (cont.) Develop high yielding biomass cultivars for cellulose conversion to bio-energy - Identify molecular markers for maturity genes Ma5 and Ma6 to be able to develop photosensitive (PS) sorghum biomass hybrids Ma5Ma5ma6ma6 is photo-insensitive (PIS) and flowers in approximately 60 days regardless of day length. ma5ma5Ma6Ma6 is photo-insensitive and flowers in approximately 60 days regardless of day length. The hybrid between these genotypes, Ma5ma5Ma6ma6 is photo-sensitive (PS) and floral initiation is only induced with day lengths less than 12h and 20min. This can be useful in both sweet sorghum and biomass sorghum in locations farther from the equator where there is more variation in day lengths (McCollum et al. reported average yield increases of 25% of PS hybrids over PIS hybrids at Amarillo, Texas).

Plant Breeding Strategies and Priorities (cont.) Develop high yielding biomass cultivars for cellulose conversion to bio-energy - Identify molecular markers for brown midrib low lignin genes genes brm-6 and brm-12 Homozygous brm-6 hybrids and brm-12 hybrids have been reported to reduce lignin content in sorghum biomass by 50%

Production Cost of alcohol from sweet sorghum in Brazil in micro-distilleries in November, 1980. Item US$ Production Cost /ha 320.00 Cost/t stalks (30 t/ha) 10.67 Cost/t stalks (40 t/ha) 8.00 Cost/t alcohol (45 liter/t and 40 t/ha) 0.22 Cost/t alcohol (59 liter/t and 40 t/ha) 0.20 Cost/t alcohol (68 liter/t and 40 t/ha) 0.17 Source: Embrapa Maize and Sorghum, Sete Lagoas, MG, Brazil. The price of ethanol today (November 5, 2007) is about US$ 0.35 L-1 at the distillery gate and US$ 0.75 to 0.90 at the pump, depending on the distance from distilleries

Incorporação de Tolerância ao Al em Cultivares de Sorgo Elite Estamos incorporando o gene para tolerância ao Al em nossas linhagens elites em seis gerações usando a casa de vegetação em dois anos. O parâmetro usado é crescimento de raiz em solução nutritiva por sete dias. Usando este tecnologia estamos empilhando genes úteis em genótipos com tolerância ao estresse múltiplo. Estamos com ensaios no campo com três níveis de Al para quantificar o efeito dos genes para tolerância ao Al tóxico na produtividade e estabilidade de produção. Seleção p/ Tolerância ao Al em Solução Nutritiva BR 007B SC 283 Tol x Suc F3 Progenies Linhagem Recorrente Fonte Susceptível x Tolerante Cross tt x TT F1 Tt BC1 tt x Tt BC1F1 ½ Tt (½ tt eliminado em solução nutritiva) BC2 tt x Tt BC2F1 ½ Tt (½ tt eliminado em solução nutritiva) . BC4F1 ½ Tt (½ tt eliminado em solução nutritiva) BC4F2 ¼ TT ½ Tt (½ tt eliminado em solução nutritiva) BC4F3 100% TT and 97% Igual ao pai recorrente Retrocruzamento Assistido para Desenvolver Linhagens de Sorgo com Tolerância ao Al Tóxico. Incorporação de Tolerância ao Al em Cultivares de Sorgo Elite e Desenvolvimento de Linhagens Isogênicos

COELHO & SCHAFFERT, 2005

Intra-Specific Diversity for Al Tolerance in Sorghum SC283 and SC566 rely on the same locus AltSB for Al tolerance Genetic basis apparently narrow 8 different Al tolerant sources x BR012 Inheritance study and linkage analysis to markers in the AltSB region SSR-based phylogenetic analysis Al tolerance gene diversity for pyramidation via breeding Stronger alleles of AltSB Different haplotypes at AltSB for candidate gene validation Foundation for future association tests

Desenvolvimento de Sorgo Mais Eficiente na Aquisição de P A identificação e conhecimento das mudanças no rhizosfero, mecanismos de aquisição e utilização de P facilitarão a manipulação destes parâmetros. Não existe conhecimento de parâmetros nos cereais correlacionados a eficiência de aquisição/utilização de P.   Grão Resposta Classe Pedigree Tol Baixa P Alta P ao P Al t/ha (%) BR 007B S 1.95 4.86 2.49 IR SC283 T 2.66 3.29 1.23 E N BR 005R 2.74 3.51 1.28 Médio Ensaio 2.16 3.60 1.43 xx Tem a necessidade de identificar ou desenvolver genótipos com diferenças na habilidade de adquirir e utilizar P eficientemente Utilizando condições de campo, solução nutritiva, e casa de vegetação, identificamos genótipos de sorgo contrastantes para aquisição e resposta ao P Plantas de Sorgo com 21 Dias em Rhizobox xxx Xxxx BR007B (IR) BR 005R (EN) SC 283 (EN) + P - P + P - P + P - P Pelos de raízes dos genótipos SC283 (eficiente) e BR007 (responsivo) em estresse de P SC283 BR007 BR007B SC 283 +P -P

Produção diferencial de Sorgoleone Sorgoleone per unit RFW (mg/g) em Sorgo SC 283 – SEM FÓSFORO Sorgoleone Production by Various Sorghum Genotypesa Sorghum Genotype Root Fresh Wt (RFW) (g) Sorgoleone mg Sorgoleone per unit RFW (mg/g) % Purity RTx433 0.15 0.10 0.67 91.8 RTx7078 1.80 12.00 98.7 RTx430 0.08 0.30 3.75 94.9 B Redlan 2.67 17.80 80.2 B Wheatland 0.33 0.83 2.50 78.3 BR 007 B – SEM FÓSFORO a- three replicates of 25 seedlings each Fonte: Chandrashekhar I, et al.; J. Agric. Food Chem. 1996, 44, 1343−1347 Photomicrograph of Sorghum bicolor roots showing a sorgoleone-rich oily exudate secreted from the root hairs (Bar=80 μm) and b closer view of a root hair with sorgoleone exuding at the tip (Bar=15 μm). Sorgoleone-rich oil from tip of root hairs also exudes from secondary roots originating either from c roots (Bar=125 μm) or d stem (adventitious roots) (Bar=350 μm) Fonte: F.E. Dayan, USDA-ARS Natural Products Utilization Research Unit, P.O. Box 8048, University, MS 38677, USA

Hibrido Sorgo Forrageiro de Corte Hibrido Sorgo Forrageiro de Corte Desenvolvimento de Cultivares de Sorgo Forrageiro de Alta Qualidade (bmr6 bmr6) Linhagens A e B bmr6 Linhagens R bmr6 Desenvolvimento de novas linhagens bmr6 A/B e R com tolerância ao múltiplo estresse A/BTx 635 A/B BR007 A/B CMSXS205 A/B CMSXS206 A/B BR008 A/B CMSXS156 A/B CMSXS 157 RTx2784 RTx2785 CMSXS912 CMSXS225 Hibrido Sorgo Forrageiro de Corte Tx635bmr6A x Tx2785bmr6R Hibrido Sorgo Forrageiro de Corte Tx635bmr6A x Tx2784bmr6R

Final Remarks Embrapa has a relative large number (50 -60) of sweet sorghum varieties available for characterization for quality and utilization in developing experimental hybrids. The variety BR501 (Brandes) is tolerant to Al toxicity and is the restorer parent in two commercial forage sorghum hybrids. Brandes probably was selected 25 years ago because it was tolerant to Al toxicity. Brandes was successfully used in two pilot sweet sorghum distilleries in Jundiai, SP and Pelotus, RS. The proof of concept of sweet sorghum as a biofuel source has been determined. The next step is an economic evaluation for competitiveness with other available raw materials. Embrapa is currently evaluating the return to an active research and development program with sweet sorghum as a source for bio-fuels.

Embrapa Maize and Sorghum Thank You Robert Schaffert Embrapa Maize and Sorghum reschaffert@hotmail.com schaffer@cnpms.embrapa.br Tel: +55-31-3779-1076

Hibrido Sorgo Forrageiro de Corte Tx635bmr6A x Tx2785bmr6R

Fiber Increases and Juice Extraction Decreases with Time Percent fiber and juive extraction of cultivar Brandes at Araras, CNPMS/EMBRAPA, 1981 and 1982 (Schaffert et al. 1986). Percent fiber and juive extraction of cultivar Wray at Araras, CNPMS/EMBRAPA, 1981 and 1982 (Schaffert et al. 1986). Fiber Increases and Juice Extraction Decreases with Time Brandes Wray

Juice Extraction Varies Between Cultivars Percent juice extraction of four sweet sorghum cultivars at CNPMS/EMBRAPA, Sete Lagoas, Brazil, 1986/87, BR 501 = Brandes; BR 505 = Wray.

Agricultural and Industrial yields of sweet sorghum in Brazil. Component Agricultural Yield Alcohol yield   (t/ha) (liter/t) (liter/ha per harvest) Stalks Range 22 - 66 55 - 85 1210 - 5610 Average 37.7 70 2639 Grain 1.4 - 6.6 310 - 370 434 - 2442 2.2 340 748 Total 1644 - 8052 3387 Source: Schaffert and Borgonovi (1980)

Mean yield of stalk, fermentable sugar, alcohol, fresh biomass and seed of sweet sorghum in experiments at the Beijing Botanical Garden Cultivar Theis M-81E Wray Keller Brandes Rio Stalk (kg/ha) 95 89 76 62 52 Fermentable sugar (t/ha) 10.6 9.6 10.3 10.5 6.4 6.2 Alcohol (l/ha) 6 159 5 607 5 981 6 131 3 696 3 617 Fresh material (t/ha) 125 128 106 107 82 Seed (kg/ha) 6 674 6 213 1 426 1 960 3 500 2 866

Sugarcane (São Paulo averages) Comparison of juice between sweet sorghum and sugarcane in Brazil. Sweet Sorghum Trait   Literature National Trials Sugarcane (São Paulo averages) Juice extraction (%) 350 - 600 500 - 700 600 - 800 Retractometer Brix 16 - 20 14 - 20. 18 -21 Sucrose (% juice) 10 -15 8 - 16. 15 - 18 Invert sugars (% juice) 1 - 4 0.7 - 7.3 0.2 - 1.5 Total invert sugars (% juice) 14 - 20 14 - 18 16 - 19 Source: Schaffert and Borgonovi (1980)

Sucrose vs. Total sugars

Maximum dry matter production and maximum growth rates of several crops. Maturity Average Growth rate Maximum growth rate Crop (t/ha) (days) (gm/m2 per day) Napier 106 365 26 ----- Sugarcane 70 18 38 sugarbeet 47 300 14 31 Forage sorghum 30 120 22 43 210 19 Sudangrass 33 160 51 Alfafa 36 250 13 23 Bermudagrass 35 230 20 Alga 44 - 74 15 - 22 28 Source: Loomis and Willian (1963)  

Location and Date of Planting National Forage Sorghum Trial - 1986/87 Embrapa Maize and Sorghum   Location and Date of Planting Linhares Goiânia Capinópolis Ituiutaba Taquari Cruz Alta S.J. dos Campos Mean Relative ES GO MG RS SP Cultivar 10.12.86 29.12.86 01.12.86 16.12.86 12.12.86 04.12.86 09.01.87 BR601 (%) Biomass Production (t/ha wet weight) BR506 (V) 46.3 55.2 43.3 55.5 41.9 59.3 50.8 52.8 109 BR507 (V) 41.1 53.2 43.7 47.3 39.8 55.7 68.1 49.5 102 BR601(H) 44.1 54.3 47.6 55.1 66.0 48.6 100 BR126* (MV) 19.1 39.6 36.6 35.0 25.5 32.3 36.0 32.1 66 Biomass Production (t/ha dry weight) 14.4 11.6 10.2 NA 13.4 17.7 18.7 14.6 108 15.6 12.4 11.7 14.1 18.9 18.2 115 12.0 12.7 14.8 18.3 14.3 13.5 7.4 16.9 15.4 9.9 11.8 11.5 85 * Forage Maize Variety * *

Percent total sugar (Juice) Wray Rio Brandes CMSXS623 Days after planting The differences between four cultivars grown in Brazil for total invert sugars of the juice during the maturity phase of production (Embrapa Maize and sorghum).

Percent fiber and juice extraction Rio a CMSXS623 Wray Brandes Days after planting The differences between four cultivars grown in Brazil for juice extraction and fiber content of the stalks during the maturity phase of production (Embrapa Maize and Sorghum). Percent fiber and juice extraction

Average yields: Stalks Leaves, peduuncle and heads Tons/ha 32.9 13.6 46.5 Estimated approximate composition of sweet sorghum sugar varieties in the United States (Nathan, 1978)

Average yields: Stalks Leaves, peduncle and heads total Tonnes/ha 47.4 19.3 66.7 Estimated approximate composition of sweet sorghum syrup varieties in the United States (Nathan, 1978)