VALUE-ADDITION TO CORN-ETHANOL STILLAGE … · STILLAGE USING OLEAGINOUS MUCOR CIRCINELLOIDES ... Biodiesel and Bio-ethanol ... " Increased efforts to remove corn oil before or after

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

Debjani Mitra Iowa State University

Lawrence Berkeley Laboratory

Corn Utilization & Technology Conference, Indianapolis 5th June, 2012

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)

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)

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)

Thin stillage

Fig. Dry-grind corn-ethanol process

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 500-600 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)

Table: Chemical characteristics

pH 4 100 COD:5N:1P

Thin stillage

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

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

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

# ATS: Autoclaved TS

* SCOD: Soluble Chemical Oxygen Demand

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)

Fungal oil – fatty acid analysis (TLC & GC)

Spent fungal biomass

Solvent addition (toluene: methanol) &

Ultrasonication (70% amplitude or 494 µmpp for 3 min)

Effluent - total solids, SCOD*, soluble sugars, acids, glycerol, nitrogen

Statistical analysis

Analysis of Variance (ANOVA) using JMP 8.0.2. - 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)

Results Fungal growth

q  Morphological state & yield of fungal biomass changed based on solid content of TS

Centrifuged thin stillage (%Total solids = 1.5 ± 0.7 %)

Whole thin stillage (% Total solids = 6 ± 2.0 %)

Biomass yield = 8 g/L Biomass yield = 22 g/L

Harvesting biomass w/ entrapped corn solids with screen

(pore size 1mm x 1mm)

Day 2 Day0 Day0 Day 2

Results Fungal lipid production

Fig: Microscopic pictures of Mucor filaments containing lipid bodies as seen under a compound light microscope 100×10 magnification

Results Fungal biomass yields

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

0

5

10

15

20

25

1 2 3 4 5

Bio

mas

s yi

eld

(g/L

) (db

)

Time (days)

Spore stock in glycerol Pellets gown in YM Pellets gown in CTS Homogenized pellets gown in YM

Results Characteristics of TS and fungal biomass

Characteristics

TS (before)

TS (after)

% Reduction

pH 4.2 ± 0.3 5.4 ± 0.3 NA

Total solids (%) 66 ± 5.0 6.6 ± 0.3 90

Total nitrogen (g/L) 5.5 ± 0.3 0.5 ± 0.04 91

SCOD (mg/L) 45.0 ± 4.0 5.0 ± 0.5 89

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 23.08 ± 1.2

Total crude fat 39.4 ± 1.5

Total protein 30.42 ± 1.0

Table: Change in thin stillage (TS) characteristics before and after fungal processing. Data are means ± SD, n=3; NA : not applicable

Results Fungal lipid extraction

Ultrasonication 70% amplitude (494 µmpp) for 3 min

Dried fungal cells in organic solvent (s)

100×10 magnification

Sonicated fungal cells in organic solvent (s)

Fungal biomass in organic solvent (s) disrupted with rotary mixer &

glass beads

Magnetic stirring After 12h

Fungal biomass in organic solvent (s) disrupted with mechanical stirring & glass beads

1. Sonication

3. Magnetic stirring 2. Rotary mixer

Results Fungal lipid yields from YM vs. TS

Parameters YM TS

Biomass yield (g/L) 5.0 ± 0.5 20 ± 0.5 #

Oil content (%) 20 ± 1.5 46 ± 2.0

Oil yield (g/L)* 1.0 ± 0.2 9.2 ± 1.0

PUFA content in fungal oil (% total lipids) 33.2 ± 1.2 51.4 ± 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)

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.

Results Fungal oil – fatty acid compositional analysis

Lipid class Lipid composition (wt. %)

Mucor-YMoil Mucor-TSoil TSoil

TAG 34.2 ± 0.7 14.7±1.8 64.5±4.3

FFA 4.2±0.5 38.4 ± 6.1 6.4±0.8

DAG 1.5±0.1 8.4±1.1 1.1±0.3

Polar 4.1±0.3 3.2±0.4 0.4±0.1

Table: Lipid class composition of oil samples Data are means ± SD, n=2

Results Fungal oil – fatty acid compositional analysis

Fatty acids Total fatty acid composition of oil

samples (wt%)

Mucor-YMoil Mucor-TSoil TSoil

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±0.4 15.7±1.0 15.0±0.2 C16:1 (Palmitoleic acid) 6.3±0.0 NA NA C18:0 (Stearic acid) 4.0±0.2 2.3±0.1 2.2±0.0 C18:1 (Oleic acid) 24.4±0.7 29.6±0.5 28.7±0.2 C18:2 (Linoleic acid) 15.7±0.2 50.0±1.6 52.5±0.2 C18:3 (Linolenic acid) 17.5±0.1 1.4±0.1 NA C20:0 (Arachidic acid) 0.6±0.0 1.2±0.1 1.5±0.1 C22:0 (Behenic acid) 0.4±0.0 NA 0.1±0.1

Table: Fatty acid composition of oil extracted from fungal cells grown on YM broth (Mucor-Ymoil) and thin stillage (Mucor-TSoil ); as well as oil from thin stillage alone (TSoil). Data are means ± SD, n=2

Conclusions

q  Mucor biomass yields of 20 g/L (db) from TS q  Optimized culture conditions

§  37 °C, pH 6.0 ± 0.5, 48 h incubation, heat sterilization, solids content - 6%, mycelial inoculum in YM

q  92% increase in oil yield from thin stillage (4.8 to 9.2 g/L) §  Additional revenue generation

q  Oil yields further increased (41%) with 10% (v/v) crude glycerol supplementation

q  High protein, high fiber fungal biomass - suitable animal feed

q  Recyclable water recovered from processed TS §  Saves energy & water

Conclusions

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

q  Future study - Select fungal strain or culture conditions - increased production of specific fatty acid, like GLA