VALUE-ADDITION TO CORN-ETHANOL STILLAGE USING OLEAGINOUS MUCOR CIRCINELLOIDES

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1 VALUE-ADDITION TO CORN-ETHANOL STILLAGE USING OLEAGINOUS MUCOR CIRCINELLOIDES Debjani Mitra Iowa State University Lawrence Berkeley Laboratory 5 th June, 2012 Corn Utilization & Technology Conference, Indianapolis

2 Acknowledgement Co authors: Mary L. Rasmussen, Priyanka Chand, Venkat Reddy Chintareddy, Linxing Yao, David Grewell, John G. Verkade, Tong Wang, and J. (Hans) van Leeuwen Agnieszka Frankowska, Miki Vincent, Carol Ziel, Bill Colonna Lincolnway Energy, IA Funding agencies: Grow Iowa Values Fund (GIVF); Iowa Energy Center (IEC); CCUR (Center for Crops Utilization Research)

3 Problems addressed/motivation q Growing importance of biofuels - Biodiesel and Bio-ethanol q Economic viability of biofuel industry Low-cost feedstock (Anoop et al., 2011) Value-addition to co-products (Antolin, 2002) q Traditional source of biodiesel - plant oils (Miao et al., 2006) Food vs. fuel controversy q Microbial lipids several advantages Do not require fertile land, have much higher yields (Meng et al., 2009) Filamentous molds are lesser explored Cellular content - 30% or more is lipids (triglycerides) (Sergeeva et al., 2008; Vicente et al., 2009)

4 Problems addressed/motivation (contd.) q Challenges in microbial biodiesel production Water and nutrient demands Organic carbon sources - ~80% of the medium costs (Li et al., 2007) q Current trend Use of low cost agro-industrial substrates Corn powder hydrolysate (Xu et al., 2006); sweet sorghum juice (Gao et al., 2010); crude glycerol (Liang et al., 2009); domestic/industrial wastewaters (Woertz, et al., 2009); shrimp waste yeast & bacterial strains (Zhang et al., 2011) q Presence of oil in growth medium increases lipid content of microbial cells Composition of intracellular lipid reflects the chemical characteristics of the oil source (Szczesna-Antczak et al., 2006) q Fungal biomass also been documented as excellent biosorbent material (Ozsoy et al., 2008) Capable of removing oil from oil water emulsions (Srinivasan and Viraraghavan, 2010)

5 Thin stillage Fig. Dry-grind corn-ethanol process

6 Thin stillage q Doubling of ethanol production (6,500 in 2007 to 13,320MGY in 2010) (Source: Renewable Fuels Association) Increase in TS production q 50 MGY plant generates gallon thin stillage/day 5 6 gal stillage/gal ethanol, half of which is recycled as backset (Rasmussen et al., in press) q TS concentration to syrup is energy-intensive. Current application as DDGS is not very economical (Moreau, 2010) q Increased efforts to remove corn oil before or after ethanol production (Majoni, 2010; Moreau, 2010) Better feed quality DDGS q Post-fermentation fractions have higher oil content especially TS (Moreau, 2010)

7 Thin stillage Table: Chemical characteristics ph COD:5N:1P

8 Hypothesis Growing Mucor circinelloides on thin stillage (TS) would increase oil yields as the fungal biomass would not only be able to adsorb and recover the corn oil from TS, also their lipolytic activity would break down the corn oil and the end products (fatty acids) used for further lipogenesis

9 Objectives Objective 1: Cultivate M. circinelloides on thin stillage (TS) & yeast-mold (YM) broth Objective 2: Optimize growth parameters highest biomass & oil yields Objective 3: Optimize oil extraction conditions Objective 4: Analyze and compare fatty acid profile of TS lipids, TS-derived fungal lipids and YM-derived fungal lipids

10 Materials and Methods Mucor cultivation Optimization of growth & oil yields Flask level studies Whole stillage vs. settled TS supernatant Sterilization method ph, temperature, %solids, incubation period Inoculum physiology spores vs. mycelia 5-L Airlift reactor study # ATS (6% solids), ph 6, 37 C, 7 SLPM aeration rate, 72 h, 10% (v/v) glycerol addition Dried biomass (proximate analysis) Solvent addition (toluene: methanol) & Ultrasonication (70% amplitude or 494 µm pp for 3 min) Effluent - total solids, SCOD *, soluble sugars, acids, glycerol, nitrogen # ATS: Autoclaved TS * SCOD: Soluble Chemical Oxygen Demand Fungal oil fatty acid analysis (TLC & GC) Spent fungal biomass

11 Statistical analysis Analysis of Variance (ANOVA) using JMP significant difference among different treatments. Least Significant Means Differences (LSD) calculated at P = 0.05 by Student s t test. All treatments were carried out in triplicates, results shown as means of three replicates ± standard deviation (SD)

12 Results Fungal growth Morphological state & yield of fungal biomass changed based on solid content of TS q Day0 Day 2 Centrifuged thin stillage (%Total solids = 1.5 ± 0.7 %) Biomass yield = 8 g/l Day0 Day 2 Whole thin stillage (% Total solids = 6 ± 2.0 %) Biomass yield = 22 g/l Harvesting biomass w/ entrapped corn solids with screen (pore size 1mm x 1mm)

13 Results Fungal lipid production Fig: Microscopic pictures of Mucor filaments containing lipid bodies as seen under a compound light microscope magnification

14 Results Fungal biomass yields 25 Spore stock in glycerol Pellets gown in CTS Pellets gown in YM Homogenized pellets gown in YM Biomass yield (g/l) (db) Time (days) Fig: Change in fungal biomass yields based on inoculum preparation. Data are means ± SD, n=3 2-L Erlenmeyer flask study, TS (total solids=4%), 37 C, 150 rpm

15 Results Characteristics of TS and fungal biomass Table: Change in thin stillage (TS) characteristics before and after fungal processing. Data are means ± SD, n=3; NA : not applicable Characteristics TS (before) TS (after) % Reduction ph 4.2 ± ± 0.3 NA Total solids (%) 66 ± ± Total nitrogen (g/l) 5.5 ± ± SCOD (mg/l) 45.0 ± ± Table: Proximate analysis of fungal biomass grown on TS. Data are means ± SD, n=2 Component (%) (db) Moisture 2.1 ± 0.1 Ash 4 ± 0.2 Total carbohydrates ± 1.2 Total crude fat 39.4 ± 1.5 Total protein ± 1.0

16 Results Fungal lipid extraction 1. Sonication Ultrasonication 70% amplitude (494 µmpp) for 3 min magnification Sonicated fungal cells in organic solvent (s) Dried fungal cells in organic solvent (s) 2. Rotary mixer 3. Magnetic stirring Magnetic stirring Fungal biomass in organic solvent (s) disrupted with rotary mixer & glass beads After 12h Fungal biomass in organic solvent (s) disrupted with mechanical stirring & glass beads

17 Results Fungal lipid yields from YM vs. TS Table: Biomass and oil yields from thin stillage (TS) and YM broth. Inoculum was grown in YM, fungal cultivation in a 6-L airlift bioreactor at 37 C, incubation period=2 days, ph = 4.5, aeration= 7SLPM. Biomass harvested by a 1mm 1mm screen. Parameters YM TS Biomass yield (g/l) 5.0 ± ± 0.5 # Oil content (%) 20 ± ± 2.0 Oil yield (g/l) * 1.0 ± ± 1.0 PUFA content in fungal oil (% total lipids) 33.2 ± ± 2.6 Data are means ± SD, n=3 # After subtracting 2 g/l corn solids (1mm 1mm screen) * TS alone had ~4.8 g/l oil (92% increase in yield)

18 Results Fungal oil fatty acid compositional analysis Table: Lipid class composition of oil samples Data are means ± SD, n=2 Lipid class Lipid composition (wt. %) Mucor-YM oil Mucor-TS oil TS oil TAG 34.2 ± ± ±4.3 FFA 4.2± ± ±0.8 DAG 1.5± ± ±0.3 Polar 4.1± ± ±0.1

19 Results Fungal oil fatty acid compositional analysis Fatty acids Total fatty acid composition of oil samples (wt%) Mucor-YM oil Mucor-TS oil TS oil C13:0 (Tridecylic acid) 12.5±1.4 NA NA C14:0 (Myristic acid) 2.3±0.1 NA NA C15:0 (Pentadecylic acid) 0.6±0.0 NA NA C16:0 (Palmitic acid) 15.8± ± ±0.2 C16:1 (Palmitoleic acid) 6.3±0.0 NA NA Table: Fatty acid composition of oil extracted from fungal cells grown on YM broth (Mucor-Ym oil ) and thin stillage (Mucor-TS oil ); as well as oil from thin stillage alone (TS oil ). Data are means ± SD, n=2 C18:0 (Stearic acid) 4.0± ± ±0.0 C18:1 (Oleic acid) 24.4± ± ±0.2 C18:2 (Linoleic acid) 15.7± ± ±0.2 C18:3 (Linolenic acid) 17.5± ±0.1 NA C20:0 (Arachidic acid) 0.6± ± ±0.1 C22:0 (Behenic acid) 0.4±0.0 NA 0.1±0.1

20 Conclusions q q q q q q Mucor biomass yields of 20 g/l (db) from TS Optimized culture conditions 37 C, ph 6.0 ± 0.5, 48 h incubation, heat sterilization, solids content - 6%, mycelial inoculum in YM 92% increase in oil yield from thin stillage (4.8 to 9.2 g/l) Additional revenue generation Oil yields further increased (41%) with 10% (v/v) crude glycerol supplementation High protein, high fiber fungal biomass - suitable animal feed Recyclable water recovered from processed TS Saves energy & water

21 Conclusions q q Advantages of fungal cultivation Heterotrophic fungal growth no dependence on light or land Filamentous nature easy and inexpensive harvest Enzymatic activity fungi can adapt to various substrates Future study - Select fungal strain or culture conditions - increased production of specific fatty acid, like GLA