Supporting information
1. Stoichiometric Yield Comparison of MixAlco Ethanol from Acetate and
Conventional Ethanol from Biomass
Fig S1 compares MixAlco and conventional ethanol production from biomass. In the
MixAlco process, the biomass is firstly fermented into acetate in the CBP using natural occurring
acid-producing microorganisms. The resulting acetate is further hydrogenated into ethanol using
hydrogen from a pipeline or refinery. In contrast, in a conventional ethanol process, the biomass
is firstly hydrolyzed to fermentable sugars, which are further directly fermented into ethanol
using recombinant microorganisms.
a) Stoichiometric Yield for MixAlco Ethanol
Fermentation
C 6 H12 O 6 (glucose)  3 CH 3 COOH (Acetate)
(S-1)
Hydrogenation
CH 3 COOH (Acetate)  2H 2  CH 3 CH 2 OH (Ethanol)  H 2 O
(S-2)
b) Stoichiometric Yield for Conventional Ethanol
Fermentation
C 6 H12 O 6 (Glucose)  2 CH 3 CH 2 OH (Ethanol)  2 CO 2
1
(S-3)
In the biomass-to-ethanol pathway of the MixAlco process, the substrate streams (e.g.,
glucose [C6]) are sent to countercurrent fermentations where the substrate is converted into
acetic acid (CH3COOH) without CO2 as a by-product. In comparison, conventional ethanol
fermentations produce one molecule of CO2 for every molecule of ethanol (CH3CH2OH).
Therefore, the carbon efficiency of MixAlco-ethanol process is nearly 100% vs. 67% [i.e., 2C in
ethanol/(2C in ethanol + 1C in CO2) = 67%] for conventional biomass-to-ethanol process.
In the MixAlco process, the mixed culture of natural microorganisms produce a wide range
of metabolic products (Holtzapple et al. 1999). As reported in this study, under controlled
conditions (i.e., controlling pH, controlling temperature, and ammonium bicarbonate buffer),
acetate content in fermentation products could reach 92% (Table 3). In contrast, pure culture
deployed in a conventional biomass-to-ethanol process can produce nearly pure (100%) ethanol.
The ratio of improved stoichiometric yield from conventional ethanol to MixAlco ethanol
can be deducted as follows:
Yield of MixAlco ethanol  Yield of convention al ethnaol
Yield of convention al ethanol

100%  92%  67% 100%
= 37.3%
67% 100%
(S-4)
In summary, the stoichiometric ethanol yield in the MixAlco process is ~40% higher than
that in a conventional biomass-to-ethanol process.
2
2. the Continuum Particle Distribution Model (CPDM)
Modeling and optimizing mixed-culture countercurrent fermentations is extremely difficult
because they involve multiple microorganisms, feedstocks, acid products, and reaction phases.
Furthermore, laboratory countercurrent fermentations are time-consuming and may take several
months to achieve steady state (Aiello-Mazzarri et al. 2005; Thanakoses et al. 2003); therefore,
optimizing a single biomass feedstock could take years. The CPDM overcomes these problems
(Chan and Holtzapple 2003).
The solid reactant in the fermentor is composed of discrete particles with differing
diameters (Fig. S2a). Generally, smaller discrete particles react more quickly, whereas larger
discrete particles react more slowly. To avoid difficulties of tracking individual discrete
particles, CPDM uses the concept of a continuum particle, which Ross defined as a collection of
particles with a VS mass of one gram when entering the fermentor (Ross 1998). By definition,
each continuum particle is identical to the others at the points of entry; however, once in the
fermentor, they can differ in the reaction extent (conversion x) (Fig. S2b).
The CPDM method characterizes the reaction rate using model parameters obtained from
batch fermentations operating at varying initial substrate concentrations (40, 70, 100, and 100+ g
dry substrate/L liquid).
The product acid concentrations can be converted to acetic acid equivalents ():
 (mol/L) = acetic (mol/L)
+ 1.75 × propionic (mol/L)
+ 2.5 × butyric (mol/L)
3
+ 3.25 × valeric (mol/L)
+ 4.0 × caprioc (mol/L)
+ 4.75 × heptanoic (mol/L)
(S-5)
On a mass basis, the acetic acid equivalent (Aceq) can be expressed as
Aceq (g/L)  60.05 (g/mol)  α (mol/L)
(S-6)
In each batch experiment, the concentrations of acetic acid equivalents were fit to
Aceq (t )  a 
bt
1  ct
(S-7)
where a, b, and c are constants, and t is the fermentation time in days. Initial values for the
parameters a, b, and c were guessed. The parameters a, b, and c were obtained by the least
square method.
The specific reaction rate ( r̂ , the reaction rate per continuum particle) is calculated by
r
rˆ 

So
d (Aceq)
dt
S0

b
(1  ct ) 2 S0
(S-8)
In a batch fermentor, the initial substrate concentration So (g VS/L liquid) is defined as the
initial volatile solid mass mo per volume of liquid V (So = mo/V). In a four-stage countercurrent
fermentation, mo is the mass of fresh volatile solids added to Fermentor 1, and V is defined as the
fresh liquid volume added to Fermentor 4 (Fig. 2b).
For each batch fermentor, the biomass conversion x is calculated by
x(t ) 
Aceq(t )  Aceq(t  0)
Soσ
(S-9)
4
where σ is the selectivity (g Aceq produced/g VS digested). In the CPDM method, the
selectivity σ is assumed constant and calculated from the selectivity s (g total acid produced/g
VS digested) by

s

(S-10)
The average value of selectivity s is determined from the countercurrent experiments. The
parameter  (the ratio of total grams of carboxylic acids to total grams of Aceq) was introduced
to avoid the inhibitory effects of higher acids that would overestimate the specific rate (Ross
1998).
Eq. S-11 is empirical and is the governing equation in the CPDM method. It relates the
specific reaction rate rˆ( x, Aceq ) with acetic acid equivalent concentration (Aceq) and fraction
conversion of volatile solids (x).
rˆpred 
e(1  x) f
1  g (  Aceq) h
(S-11)
where e, f, g, and h are empirical constants. The (1 – x) term was described as the conversion
penalty function (South and Lynd 1994). The least square method was used to determine the
empirical constants e, f, g, and h. The resulting Aceq was converted back to total carboxylic acid
concentration using parameter .
5
References
Aiello-Mazzarri C, Coward-Kelly G, Agbogbo FK, Holtzapple MT. 2005. Conversion of
municipal solid waste into carboxylic acids by anaerobic countercurrent fermentation Effect of using intermediate lime treatment. Applied Biochemistry and Biotechnology
127(2):79-93.
Chan WN, Holtzapple MT. 2003. Conversion of municipal solid wastes to carboxylic acids by
thermophilic fermentation. Applied Biochemistry and Biotechnology 111(2):93-112.
Holtzapple MT, Davison RR, Ross MK, Aldrett-Lee S, Nagwani M, Lee CM, Lee C, Adelson S,
Kaar W, Gaskin D and others. 1999. Biomass conversion to mixed alcohol fuels using the
MixAlco process. Applied Biochemistry and Biotechnology 77-9:609-631.
Ross MK. 1998. Production of Acetic Acid from Waste Biomass [Ph.D. dissertation]. College
station, TX: Texas A&M University.
South CR, Lynd LR. 1994. Analysis of Conversion of Particulate Biomass to Ethanol in
Continuous Solids Retaining and Cascade Bioreactors. Applied Biochemistry and
Biotechnology 45-6:467-481.
Thanakoses P, Black AS, Holtzapple MT. 2003. Fermentation of corn stover to carboxylic acids.
Biotechnology and Bioengineering 83(2):191-200.
6
Figures Caption
Fig. S1. Routes to converting biomass to ethanol.
Fig. S2. Overview of continuum particle in the CPDM method. a) Definition of continuum
particle; b) reactant conversion from a continuum particle.
Fig. S3. Routes to fuels and chemicals in downstream processing starting from carboxylate salts.
End products of the MixAlco process are mixed primary alcohols (e.g., ethanol), mixed
secondary alcohols (e.g., isopropanol), and hydrocarbons (e.g., alkanes). The intermediate
products (e.g., acetic acid) are valuable chemicals and could be sold as desired products.
Fig. S4. Pretreatment devices used to treat sugarcane bagasse with aqueous ammonia. a)
Modified temperature-adjustable oven; b) Self-constructed high-pressure reactors; c) Roller
system; d) 1-L centrifuge bottle. Both a) and b) are for short-time ammonia treatment, whereas
c) and d) are for long-term ammonia treatment.
7
Sugars
Conventional ethanol
Pure-culture
fermentation
Substrate
hydrolysis
Biomass
Ethanol
Acetate
MixAlco ethanol
Mixed-culture
fermentation
Hydrogeneration
Fig. S1.
8
Fig. S2.
9
Carboxylate
salt
Ac
id
sp
rin
gin
g
Carboxylic
acid
Ketone
Ester
H2
H2
Secondary
alcohol
Ol
(ze igom
oli eri
te
cat zatio
aly n
st)
n
tio )
iza st
er ly
m ata
igo te c
Ol eoli
(z
Oligomerization
(zeolite catalyst)
Ether
Hydrocarbons
Fig. S3.
10
Primary
alcohol
Fig. S4.
11