Process Biochemistry 51 (2016) 10461057

Contents lists available at ScienceDirect

Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Review

Soybean carbohydrate as fermentation feedstock for production of

biofuels and value-added chemicals
Abdullah Al Loman, Lu-Kwang Ju
Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, OH 44325-3906, USA

a r t i c l e i n f o a b s t r a c t

Article history: Being one of the major oilseed crops throughout America and Asia, soybean production has increased
Received 5 March 2016 rapidly due to the rising demand for oil and protein. Carbohydrates are also major components in soybean.
Received in revised form 5 April 2016 During the soybean processing for producing oil and protein products, large amounts of carbohydrate-rich
Accepted 16 April 2016
byproducts or waste are generated. Soy protein products such as soybean meal, soy protein concentrate
Available online 19 April 2016
and soybean milk also contain carbohydrates that have anti-nutritional concerns and decrease the value
of these products. Removing these carbohydrates from soy protein products and finding valuable uses
Keywords:
for these carbohydrates and other carbohydrate-rich waste/byproducts are highly desirable. Here, the
Soy carbohydrate
Biofuel
various carbohydrate-rich waste byproducts generated from soybean processing and their compositions
Enzyme are described. Recent developments on their use as fermentation feedstocks for production of biofu-
Fermentation els, enzymes and a variety of specialty chemicals are then reviewed and summarized. This review can
Bio-refinery facilitate knowledge and technology integration for development of a soy-based biorefinery platform.
2016 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1047
2. Current soybean processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1047
2.1. Overview of current processing and derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1047
2.1.1. Soybean hull . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1047
2.1.2. Soybean meal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1047
2.1.3. Soybean molasses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1048
2.1.4. Soybean okara . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1048
3. Biofuel production using soybean carbohydrate as fermentation feedstock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1048
3.1. Ethanol production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1048
3.1.1. Ethanol from soybean hull hydrolysate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1048
3.1.2. Ethanol from soybean molasses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1050
3.2. Butanol production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1050
4. Enzyme production from soybean processing byproducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1051
4.1. Cellulase production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1051
4.2. Polygalacturonase production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1051
4.3. Xylanase production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1051
4.4. Lipase production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1051
5. Specialty chemical production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1053
5.1. Specialty chemicals from soybean meal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1053
5.2. Specialty chemicals from soybean molasses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1053
5.3. Specialty chemicals from soybean okara . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1053
5.3.1. Methane production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1053

Corresponding author.
E-mail address: Lukeju@uakron.edu (L.-K. Ju).

http://dx.doi.org/10.1016/j.procbio.2016.04.011
1359-5113/ 2016 Elsevier Ltd. All rights reserved.
 A.A. Loman, L.-K. Ju / Process Biochemistry 51 (2016) 10461057 1047

5.3.2. Hydrogen production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1054

5.3.3. Antibiotic production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1054
5.3.4. Improving antioxidant properties of polysaccharide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1054
6. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1054
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1055
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1055

1. Introduction Soybean hulls are first removed. The dehulled flakes are then
pressed or solvent-extracted for oil, leaving the soybean meal that
The ability of soy to grow in a wide variety of soil and climatic contains a high percentage of protein [21]. Soybean meal can be
conditions makes it a versatile crop and one of the most widely used as a source of protein supplements in animal feed and food.
grown oilseed crops. Besides production of vegetable oil for human To further raise the protein content of soybean meal, water (with
consumption, soys protein-rich meal has high value as functional or without ethanol) extraction of soluble sugar at pH around the
and nutritional ingredients for food and feed uses. Oil and protein isoelectric point of soy protein is done to produce the soy protein
together make up approximately 60% of the soybean weight; car- concentrate [22]. Alternatively, soy protein isolate can be produced
bohydrate, 2630%, is another major component [1]. During the by dissolving protein at high pH, separating the solution from
processing of soybeans for making the oil and protein products, remaining solids, and then reprecipitating protein from the solu-
large amounts of byproducts or waste are also generated, which tion by lowering pH to the isoelectric point [23]. Soybean okara is
are rich in carbohydrates [2]. The large amounts of soybean carbo- the solid remainder collected after most protein is made soluble,
hydrates, mainly ended up in these waste or low-value byproducts, e.g., in making either soy protein isolate or soybean milk [24]. Soy-
are yet to find high-value applications [3]. These waste/byproducts bean okara contains mainly insoluble polysaccharides. On the other
include the hulls separated from beans before oil extraction [4], hand, soy molasses contains the soluble carbohydrate collected in
molasses generated from the production of soy protein concentrate the aqueous streams during the making of soy protein concen-
(SPC) [5], and soybean okara created during production of soy pro- trate and isolate [15]. The principal carbohydrate-rich byproducts
tein isolate (SPI) and soybean milk, respectively [6]. A flow diagram from soybean processing include hulls, meal, okara and molasses.
is given in Fig. 1 for the various products made by current soybean Except soybean meal, they are yet to find valuable large-scale
processing. More detailed description is given in Section 2.1. industrial applications. Their compositions and/or characteristics
Soybean meal, a protein-rich product mainly used as animal are described in the following sections.
feed, also contains large proportions of carbohydrates. Majority
of the carbohydrate are structural polysaccharides and galactose- 2.1.1. Soybean hull
containing oligosaccharides such as stachyose and raffinose, which Soybean hull represents almost 810% of the whole soybean
the animal may lack the necessary endogenous enzymes to digest [13]. Considered as having much less commercial value than the
[7]. Their presence reduces the nutritional value of soybean meal for oil and protein, hulls have not received much attention beyond the
use in animal feed. Removal of undesirable carbohydrates not only use as animal feed. They are typically sold as is or as compressed
can improve the digestibility but also increase the protein content pellets and fed to cattle and pigs. Soybean hull contains mainly
of soybean meal [811]. The carbohydrates separated from soybean cell wall polysaccharides. It has a relatively small amount of lignin
meal may also be used for the production of value-added chemicals (14%) compared to other agricultural residues. Lignin is a major
and fuels [1215]. hindrance for the hydrolysis of many other lignocellulosic materials
Fermentation can be used to convert soybean carbohydrates, to fermentable sugar. The low lignin content makes soybean hulls
either present in carbohydrate-rich waste/byproducts or separated potentially an easier feedstock for production of biomass-derived
from soybean meal, to more valuable bio-products. Many fermenta- chemicals. Composition of soybean hulls on a dry basis is presented
tion processes require or strongly favor the use of monosaccharides in Table 1. The relatively wide content ranges of different com-
as substrate. For these processes, pretreatment and hydrolysis for ponents reflect the dependency of nutritional value and chemical
carbohydrate monomerization are done before or during the fer- composition of soybean hulls on the processing nature and growing
mentation; the latter is termed simultaneous saccharification and conditions (demographic and seasonal).
fermentation. Some microorganisms can utilize complex soy car-
bohydrates, containing oligo- and poly-saccharides, directly and 2.1.2. Soybean meal
are often investigated in solid-state fermentation [1619]. These Soybean meal is an abundant byproduct after oil extraction. It
fermentation processes using soybean carbohydrate as major feed- has become an increasingly more important source of protein in
stock to produce value-added bio-products have been an active animal diets, particularly for poultry, swine, cattle and fish [25]. It
area of research but have not been systematically reviewed. In this is by far the dominant protein supplement used in livestock and
work, an overview is first given for the current soybean process-
ing and the various waste and byproducts generated. Production of
Table 1
biofuels (ethanol and butanol), commercially important enzymes Compositions of soybean hulls, meal, okara and molasses on a dry basis [116120].
and specialty chemicals are then described in more detail.
Components Composition (weight%)

Hulls Meal Okara Molasses

2. Current soybean processing
Cellulose 2951 24 1015
2.1. Overview of current processing and derivatives Hemicellulose 1020 810 512
Soluble oligosaccharides 510 1012 25 5560
Lignin 14 0.51 12
A flow diagram is given in Fig. 1 for the various products Pectin 615 1012 2535
made by current soybean processing. Recovery of soybean oil is Protein 914 5052 2530 812
done by physical and chemical methods such as hard screw press- Ash 14 56 45 57
Fat 23 37 1520
ing, prepress-solvent extraction and direct solvent extraction [20].
1048 A.A. Loman, L.-K. Ju / Process Biochemistry 51 (2016) 10461057
Fig. 1. Current soybean processing [21,114,115].
poultry feeds in the United States and throughout the world. Soy-
bean meal has an excellent profile of essential amino acids that
meets all the requirements of animal protein supplements and
the digestibility of protein is comparable to the commonly avail-
able protein sources [26]. Soybean meal also contains a significant
amount of carbohydrates. A large fraction of the carbohydrates
is, however, insoluble and/or poorly digestible. For use in animal
feeds, it is desirable to degrade the anti-nutritional polysaccha-
rides and oligosaccharides in the meal. The composition of soybean
meal may vary depending on the origin and cultivar of soybean,
environmental growing conditions, and processing conditions. On
the wet weight basis, soybean meal consists of 4850% protein,
3032% carbohydrate, 56% ash and 23% fat [27]. Composition of
the carbohydrate components is given in Table 1.
2.1.3. Soybean molasses
Soybean molasses is brown viscous syrup with a bittersweet
flavor. It is generated during the processing for production of soy
protein concentrate and soy protein isolate. The solvent used in
the processing dissolves substances from soybean meal; molasses
is then formed after desolventization [15]. Soybean molasses is
mainly composed of soluble carbohydrates, protein (including pep-
tides and amino acids), lipid (fat) and ash (Table 1). The most
abundant carbohydrate components are sucrose (28%), stachyose
(18%) and raffinose (9%) [28].
2.1.4. Soybean okara
Okara is the fiber-rich residue left from ground soybeans after
the water soluble fraction is extracted for production of soybean
milk, tofu, fried bean curd, soy protein isolate, and other protein-
rich food. About 1.1 kg okara is collected per kg soybean milk
produced. Okara has been reported to have health benefits such as
an anti-cholesterol effect [29] and prevention of liver fat accumu-
lation [30,31]. Still, it is typically considered as agricultural waste
because of the difficulty for its high-volume utilization. The dif-
ficulty lies mainly in the digestion and solubilization of the high
content of complex polysaccharides. The large quantity of okara
generated and the high cost associated with its elimination cause
economic and environmental problems. Searching for commercial
uses of soybean okara has received much attention. While varying
with the actual processing involved, okara contains mainly polysac-
charides (5055%), protein (2530%), and fat (37%) [32].
3. Biofuel production using soybean carbohydrate as
fermentation feedstock
Renewable fermentation feedstocks have attracted due atten-
tion in recent years. Soybean carbohydrates are not yet studied
as extensively as other lignocellulosic feedstocks for use in fer-
mentation. Recent studies on using soybean carbohydrates or
their hydrolysate for biofuel and specialty chemical production
are reviewed here. Hydrolysis methods and conditions for various
soybean-derived materials and their hydrolysis outcomes such as
sugar yield, degree of monomerization, and inhibitory compound
generation vary extensively. They are not included in this review
so that the focus can be kept on the fermentation processes for pro-
ducing bioproducts from soybean carbohydrates. A separate review
on that topic is warranted.
3.1. Ethanol production
Among soybean by-products, the hull hydrolysate has highest
carbohydrate content and is the substrate studied most for ethanol
fermentation. Hydrolyzed soybean molasses and okara hydrolysate
[33] have also been investigated.
3.1.1. Ethanol from soybean hull hydrolysate
As given in Table 1, soybean hull contains high fractions of
polysaccharides, including 2951% cellulose, 1020% hemicellu-
lose and 615% pectin [13]. Their degrees of monomerization in
 A.A. Loman, L.-K. Ju / Process Biochemistry 51 (2016) 10461057
Fig. 2. Summary of fermentation products made from soybean processing byproducts.
the hydrolysate depend on the hydrolysis methods and condi-
tions as mentioned earlier. Monomerized sugars in hydrolysate
may be fermented to ethanol. Ethanol can also be produced
from partially hydrolyzed hulls by simultaneous saccharification
and fermentation (SSF). The composition of hull hydrolysate and
presence of oligosaccharides and toxic compounds produced by
pretreatment methods have profound effects on the ethanol pro-
duction efficiency and overall yield in the fermentation process. The
techniques that have been used to improve the ethanol production
are reviewed in the following sections.
3.1.1.1. Co-culture fermentation. Presence of both hexose and
pentose in soybean hull hydrolysate makes the fermentation chal-
lenging because majority of the organisms cannot simultaneously
metabolize them. Few microorganisms can use xylose as a sole car-
bon source. Additionally, the presence of glucose can negatively
affect pentose metabolism by catabolite repression of required
enzymes or by inactivation of enzymes in the high affinity trans-
port system [34]. Co-culture is a possible approach. For example,
co-culturing Saccharomyces cerevisiae with Candida shehatae or
Spathaspora arborariae was investigated [35]. The mixed cultures
produced ethanol from glucose and xylitol from xylose. The ethanol
1049
yield was high, up to 0.48 g/g glucose; the xylitol yield of 0.25 g/g
xylose was less satisfactory. The low xylitol yield was because the
operating condition used was not optimal for xylitol production.
3.1.1.2. Simultaneous saccharification and fermentation (SSF). SSF is
often used to ferment substrate that requires degradation before
becoming metabolizable to the microorganisms [3638]. This pro-
cess reduces the pretreatment requirement before fermentation.
SSF by S. cerevisiae and Scheffersomyces stipites and a commercial
enzyme mixture including cellulase, !-glucosidase and pectinase
was investigated on different soybean substrates, such as meal,
flakes and hulls [39]. The solid substrates were not pretreated or
sterilized; liquid was passed through a 0.2-"m filter before being
added to the SSF reactor. All these substrates supported ethanol
production in this process, up to 23 g/l from 10% (w/v) solid loading.
In another study, Mielenz et al. [13] reported using a SSF process to
ferment soybean hulls to ethanol by S. cerevisiae without any pre-
treatment. When an enzyme mixture of cellulase, !-glucosidase
and pectinase was added with 15% hulls, 2530 g/l ethanol was
produced.
1050 A.A. Loman, L.-K. Ju / Process Biochemistry 51 (2016) 10461057
3.1.1.3. Fermentation with hydrolysate containing high levels of
inhibitory compounds. Acid hydrolyzed soybean carbohydrate
hydrolysates can contain inhibitory compounds such as acetic
acid, furfural and hydroxymethylfurfural (HMF). The concentra-
tions of inhibitory compounds depend on the composition of
lignin, cellulose crystallinity, and strength and type of acid used
for hydrolysis [40]. Soybean hull has much lower lignin content
(only 14%, Table 1) than other lignocellulosic biomass such as
hardwood (1825%), softwood (2535%), corn cobs (15%), and
wheat straw (15%) [41]. Thus comparatively milder pretreatment
is often sufficient for soybean hull hydrolysis and the resultant
inhibitory compound concentrations are lower than those in other
lignocellulosic hydrolysates, where acetic acid, furfural and HMF
concentrations can be as high as 6, 1.2 and 3.5 g/l, respectively
[42]. For example, Hickert et al. [35] hydrolyzed soybean hull
with 1% sulfuric acid at a solid-to-liquid ratio of 1:20 and found
the hydrolysate to contain 2.1 g/l acetic acid, 0.08 g/l furfural,
and 0.58 g/l HMF. However, the hydrolysate is often concentrated
to achieve a higher sugar concentration, which would increase
the inhibitory compound concentrations as well. The inhibitory
concentration thresholds of inhibitory compounds also differ for
different microorganisms. Felipe et al. reported that acetic acid at
higher that 3 g/l concentrations was toxic for Candida guilliermondii
[43]. S. cerevisiae is known for having higher tolerance, which makes
it an attractive option for ethanol production [44,45]. Lee et al. [46]
reported that S. cerevisiae could tolerate up to 5 g/l acetic acid with-
out noticeable inhibition to cell growth or ethanol production. The
growth and ethanol production of S. cerevisiae [47] were reported to
be inhibited by 5 g/l furfural. However, Hickert et al. [35] found that
a soybean hull hydrolysate with 0.58 g/l HMF, 0.08 g/l furfural and
2.1 g/l acetic acid was inhibitory to S. cerevisiae. They concluded that
although these concentrations were below the inhibitory levels, the
growth and ethanol production were affected by the high osmotic
pressure (2950 mOsm/kg) of the hydrolysate. They suggested that
osmolality is also an important factor to consider. In the following
section, the reported efforts to neutralize the toxic compounds for
ethanol fermentation are summarized.
3.1.1.4. Hydrolysate detoxification. When inhibitory compound
concentrations are high, it is difficult to use the hydrolysate for fer-
mentation. Sometimes the hydrolysate requires concentrating to
increase the carbohydrate concentration but this process also ele-
vates the inhibitory compound concentrations. Activated carbon
may be used to detoxify the hydrolysate. Adsorption by 2% acti-
vated carbon at pH 2 and 80 C for 60 min removed the inhibitory
formic acid (1.45 0.08 g/l) and ferulic acid (0.32 0.07 g/l) from a
vacuum-concentrated soybean hull hydrolysate that had been pre-
pared by acid (0.1 N HCl) hydrolysis [48]. To another soybean hull
hydrolysate, which had also been vacuum-concentrated to increase
the sugar concentration, Schirmer-Michel used activated charcoal
to minimize the toxic compounds. Charcoal was mixed with soy
hull hydrolysate at 10% w/v concentration at pH 2.5 for 1 h and then
the pH of treated hydrolysate was adjusted to 5.5 to precipitate fur-
fural and phenol and, thus, reduce their dissolved concentrations
in hydrolysate by 89% and 29%, respectively [49].
3.1.1.5. Cell immobilization. As previously described, acid
hydrolyzed or pretreated soybean hull hydrolysates typically
have high concentrations of inhibitory compounds such as fur-
fural, HMF and acetic acid and high osmotic pressures, which cause
negative effects to yeast growth and ethanol production [50]. To
overcome this problem, several researchers used immobilized
cells in fermentation. Free S. cerevisiae cells could not grow well in
a medium containing 5 g/l furfural, whereas the immobilized cells
grew without a lag phase [47]. In another study, immobilized S.
cerevisiae cells were found to grow and produce ethanol at a yield
of 0.43 g/g glucose equivalent from a soybean hull hydrolysate with
approximately 3000 mOsm/kg osmolality, which was inhibitory
for free-cell fermentation [35].
3.1.2. Ethanol from soybean molasses
Soybean molasses contains soluble carbohydrates. Attempts
have been made to produce ethanol from it [15,51]. Yeast typi-
cally cannot metabolize all the carbohydrates present. They cannot
hydrolyze oligosaccharides such as stachyose and raffinose to
fermentable sugars [51]. So the ethanol yield with yeast fermen-
tation is not high enough. Ethanol yields were often given in
percentages of the theoretical yield from consumed carbohy-
drate (as the% values cited in the following reports) while large
fractions of the carbohydrate were not consumed. For example,
Machado [52] reported a 55% ethanol yield using S. cerevisiae fer-
mentation at pH 6 and 30 C. In another study, 12.5 g/l ethanol was
produced from 240 g/l soy molasses by S. cerevisiae fermentation,
while 125 g/l carbohydrate was left unconsumed in the medium
[53]. Silva et al. also studied the ethanol production from soy-
bean molasses by S. cerevisiae fermentation. They reported a 10.4%
improvement in ethanol yield from #-galactosidase pretreated
molasses, over the 72% yield achieved without the enzymatic pre-
treatment [54]. The bacterium Zymomonus mobilis is known to
provide several advantages over yeast for ethanol fermentation,
such as higher glucose uptake rate, theoretical ethanol yield (via
Enter-Doudoroff pathway [55]) and ethanol tolerance [5660].
Letti et al. [51] investigated the ethanol production from soy-
bean molasses by Z. mobilis and obtained up to 29.3 g/l ethanol
from 200 g/l molasses. This corresponded to 96% of the theoret-
ical ethanol yield from consumed carbohydrate, higher than the
89% yield obtained by using S. cerevisiae. Z. mobilis was found to
degrade stachyose to raffinose but not raffinose or melibiose; as a
result, raffinose concentration increased with fermentation time in
the study.
3.2. Butanol production
Butanol has also been considered as an alternative fuel. Bio-
based butanol is mainly produced via acetone-butanol-ethanol
(ABE) fermentation by solvenogenic clostridia such as Clostridium
acetobutylicum [61] and Clostridium beijerinckii [62]. ABE fermenta-
tion to produce butanol has been investigated utilizing different
lignocellulosic biomass where the presence of both hexose and
pentose sugars in the hydrolysate is again a challenge [63]. The
same challenge applies to butanol production from hydrolysate of
soybean carbohydrate. Recently Yu et al. [64] used a recombinant
Clostridium tyrobutyricum to simultaneously consume glucose and
xylose in an acid hydrolyzed soybean hull hydrolysate for butanol
production. For use in the fermentation medium, the hydrolysate
was concentrated three times to increase the total sugar con-
centration to 70 g/l. Similar to the effects on ethanol-producing
yeast, some compounds produced from sugar degradation by the
acid hydrolysis process, such as furfural, HMF and formic acid,
are inhibitory to the Clostridium species and can negatively affect
butanol production. It was found that furfural, HMF and formic acid
at concentrations lower than 1.0 g/l were not inhibitory to the ABE
fermentation. On the other hand, ferulic acid and p-coumaric acid
generated from lignin degradation were much more toxic to the
C. tyrobutyricum strain. They inhibited the ABE fermentation even
at 0.25 g/l. The 3-fold concentrated soybean hull hydrolysate was
found to contain 1.45 g/l formic acid and 0.32 g/l ferulic acid, which
were higher than their inhibitory thresholds. The hydrolysate was
therefore detoxified by activated carbon adsorption using 2% w/w
activated carbon at pH 2.0 and 80 C for 60 min. Using the detoxified
hydrolysate, they obtained 15.7 g/l butanol with 0.24 g/g biomass
yield and 0.29 g/l-h productivity.
 A.A. Loman, L.-K. Ju / Process Biochemistry 51 (2016) 10461057
In another study, C. acetobutylicium ATCC 55025 was found to
be more effective than three other clostridia tested, i.e., C. ace-
tobutylicum ATCC 824, C. beijerinckii BA101, and C. tyrobutyricum
(!ack, adhE2), in ABE production from 54 g/l soybean molasses
sugars [48]. In 72 h, they produced up to 15 g/l ABE including maxi-
mally 8.7 g/l butanol. They hypothesized that higher concentrations
could be obtained by using higher sugar concentrations and longer
fermentation time. Another study reported ABE production from
spray dried soybean molasses (SDSM) by C. beijerinckii BA101 fer-
mentation. SDSM contains 747 g/kg of total sugars and 434 g/kg
were fermentable by C. beijerinckii BA101. Their study suggested
that the high salt concentration (10 g/l NaCl) in molasses inhibited
cell growth and butanol yield if used at more than 80 g/l SDSM
[65]. From the maximal level of 80 g/l SDSM, C. beijerinckii BA101
produced 10.7 g/l ABE. ABE concentration was improved to 30.1 g/l
when 80 g/l SDSM was supplemented with 25.3 g/l glucose.
4. Enzyme production from soybean processing byproducts
There has been significant research on conversion of lignocel-
lulosic biomass to biofuels and chemicals. To achieve this goal of
biorefinery, biomass-degrading enzymes play a significant role in
degrading the complex biomass to fermentable sugar. Enzymes are
one of the major costs for biorefinery processes and these large-
scale processes require large amounts of enzymes. The enzyme
composition is another important factor as each biomass mate-
rial requires a specific enzyme mixture with the proper ratio of
activities that may include cellulase, hemicellulase, pectinase, #-
galactosidase and other accessory enzymes. To decrease the cost of
the optimal enzyme mixture, an effective but inexpensive induc-
ing substrate is highly desirable. The carbohydrate-rich soybean
processing waste/byproducts can serve as the inexpensive sub-
strate for production of various biomass-degrading enzymes. There
have been several such attempts for enzyme production from
soybean-based substrates, using different microorganisms in both
solid-state and submerged fermentation processes. A summary is
given in Table 2. Process conditions considered for production of
individual enzymes are described in the following sections.
4.1. Cellulase production
Filamentous fungi such as Aspergillus and Trichoderma species
are well-known effective producers of cellulase. Soybean process-
ing byproducts have been investigated as substrates for cellulase
production. In a solid state fermentation with non-pretreated okara
alone (no other supplements), effective production of cellulase with
high cellobiase (!-glucosidase) activities was achieved with B. sub-
tilis Pa5 [66]. The fermentation was done at 30 C for 48 h using
10 g okara as substrate in a 250 ml flask with initial moisture con-
tent of 71.5%. Inoculation was at a density of 1 106 CFU/ml with
1 ml inoculum. Maximum activities of cellulase and its cellobiase
component obtained were 48.4 FPU (filter paper unit) and 154.5
IU, respectively, per g dry okara used. This cellulase activity is sig-
nificantly higher compared to the reported cellulase and cellobiase
activity of 2.8 FPA and 4.5 IU per g of dry solid produced by Bacil-
lus subtilis CBTK 106 using banana stalk waste as substrate [67].
They reported that additional nutrients were not required to stimu-
late faster cell growth because small quantities of soluble nutrients
were already present in the okara [68].
Among soybean processing byproducts, soybean hull has the
highest cellulose content, which is potentially good for cellulase
production. Brijwani et al. studied cellulase production by solid
state fermentation at 30 C for 4 days using soybean hull and wheat
bran at a 4:1 ratio as the substrate [69]. The initial moisture con-
tent was 70%. By monoculture of T. reesei and Aspergillus oryzae,
1051
respectively, the cellulase and cellobiase activities obtained, in U
per g dry substrate used, were 6.5 and 6.3 for the former and 6.7
and 9.4 for the latter. By coculturing two species in the solid state
fermentation, they improved the production of both cellulase and
cellobiase to 10.7 U/g.
4.2. Polygalacturonase production
Soybean based substrate was found to induce polygalacturonase
expression. Coffman et al. [70] studied the submerged fermenta-
tion of T. reesei Rut C-30 for carbohydrase production to degrade
carbohydrate in soybean meal. Using 20 g/l soybean hull as sub-
strate, 8.0 U/ml polygalacturonase was produced (together with 3.2
FPU/ml cellulase and 248 U/ml xylanase), compared favorably to
the 1.4 U/ml polygalacturonase produced with Avicel as substrate
(together with 3.6 FPU/ml cellulase and 171 U/ml xylanase). They
also compared the polygalacturonase production using soybean
flour versus urea-proteose peptone as the nitrogen source (at equal
total N concentrations), with Avicel as the carbon substrate. The use
of soy flour increased the polygalacturonase activity to 3.8 U/ml
from 1.4 U/ml.
4.3. Xylanase production
As mentioned earlier, high xylanase production (248 U/ml) by
T. reesei Rut C-30 using soybean hull as substrate was observed by
Coffman et al. [70]. Xylanase production by solid state fermentation
was also studied using soybean based substrate. Maciel et al. [71]
studied the xylanase production by A. niger LPB 326 using soybean
meal and/or sugarcane bagasse as substrate. The solid state fermen-
tation was carried out at 85% moisture content and 30 C for 4 days.
They evaluated the effect of substrate composition, i.e., the ratio
of sugarcane bagasse to soybean meal. At the ratio of 1.86:1, i.e.,
65% sugarcane bagasse and 35% soybean meal, the xylanase activ-
ity was the highest, at 3099 IU/g dry substrate, much higher than
the only 20 U/g activity achieved with sugarcane bagasse alone. This
xylanase activity was also significantly higher than those reported
when using other substrates such as wheat bran (1024 U/g) [72]
and wheat straw (29.8 U/g) [73]. Xylanase was also produced from
soybean okara by the solid state fermentation of Bacillus circulans
BL53 at 37 C [74]. The maximal xylanase activity achieved after
72 h was 950 IU/g of protein, using 18.8 g dry okara with initial
moisture content of 86%. The xylanase produced was reported to
have very high thermal tolerance, up to 88 C. Optimal activity was
found at 80 C and pH 7.
4.4. Lipase production
Lipase is an important enzyme used in various industrial appli-
cations, for hydrolysis of ester bonds or catalysis of esterification
and transesterification. Effective lipase production can be obtained
using soybean meal as substrate. Vargas et al. [75] reported the
lipase production from soybean meal by solid state fermentation of
Penicillium simplicissimum at 27.5 C for 7 days. The initial moisture
content was 55%. They investigated the effects of supplementing
soybean meal with different carbon and nitrogen sources, including
wastewater from swine slaughterhouse, yeast hydrolysate, soy-
bean oil and corn steep liquor. Soybean meal alone was found to
be the best substrate for the lipase production, giving a maximum
lipase activity of 30 U per g dry substrate. Rigo et al. [14] reported
significantly higher lipase production by solid state fermentation
of Penicillium P58 and P74 using soybean meal supplemented with
1 g soybean oil and 3 g urea per 100 g soybean meal. The highest
lipase activity achieved after 72 h was 180 U per g dry substrate.
1052 A.A. Loman, L.-K. Ju / Process Biochemistry 51 (2016) 10461057

Table 2
Enzymes produced using soybean processing byproducts as fermentation substrate.

Strain Enzyme Soy substrate Conditions Maximum activity Ref

T. reesei & Aspergillus Cellulase, !-glucosidase & Hull Solid state co-culture: 30 C, 70% Cellulase: 10.7 FPU, !-glucosidase: [69]
oryzae xylanase moisture, pH 5, 96 h; with wheat bran 10.7 IU & xylanase: 515 U (/g dry
at 25% of hull substrate)
Bacillus subtilis Pa5 Cellulase & cellobiase Okara Solid state fermentation: 30 C, 71.5% Cellulase: 48.4 0.9 FPU & cellobiase: [66]
(!-glucosidase) moisture, 48 h, inoculum: 1 104 154 2 IU (/g dry okara)
cells/g okara
A. niger P47C3, Cellulase, !-glucosidase & Hull (bran) Solid state fermentation: 37 C, 50% Cellulase: 5.6 FPU, xylanase: 484.2 IU & [121]
Aspergillus fumigatus xylanase moisture, 120 h !-glucosidase: 6.5 IU (/g dry substrate)
P40M2
A. niger Cellulase & xylanase Meal Solid state fermentation: 32 C, 84% Cellulase: 0.55 IU, endogluconase: 35.1 [122]
moisture, 70% inlet air humidity, 96 h IU & xylanase: 47.7 IU (/g solid
substrate)
T. reesei Poly-galacturonase, xylanase & Hull, meal Submerged fermentation: 20 C, 20%+ Polygalacturonase: 11.6 U (pH 4.04.5) [70]
cellulase dissolved oxygen concentration, pH or 8.0 U (pH 6), xylanase: 248.9 U &
46 cellulase: 3.6 FPU (both @ pH 6), per ml
broth
A. niger LPB 326 Xylanase Meal Solid state fermentation: 30 C, 85% 3099 IU/g dry substrate [71]
moisture, 96 h; 35% meal + 65%
sugarcane bagasse
Bacillus circulans BL53 Xylanase (thermo-tolerant: Okara Solid state fermentation: 37 C, 86% 950 U/g of protein at 80 C (optimal) [74]
5088 C) moisture, pH 47, 72 h
A. niger NRRL 328 Xylanase Hull Solid state fermentation: 28 C, 70% 950 U/g dry substrate [123]
moisture, 168 h
Penicillium P58, P74 Lipase Meal Solid state fermentation: 27 C, 55% 146.8 U/g dry substrate [14]
moisture, 48 h; with 1 g oil & 3 g urea
per 100 g meal
Penicillium Lipase Meal Solid state fermentation: 27.5 C, 55% 30 U/g dry substrate [75]
simplicissimum moisture, 48 h
Lactobacillus agilis LPB #-galactosidase Vinasse Submerged fermentation: 30 C, pH 11.1 U/ml [124]
56 6.5, 144 h; vinasse with 30% soluble
Aspergillus japonicus !-fructo-furanosidase Okara Submerged fermentation: 30 C, pH 180 U/ml [111]
6.57, 48 h; with 3% okara & 15%
sucrose

Table 3
Bioproducts obtainable by fermentation using okara as substrate.

Strain Product Conditions Maximum yield Ref.

Anaerobic digestion Methane Anaerobic digestion: 36 C, 90% Methane: 478495 ml/g volatile solids, i.e., [18]
sludge moisture, 0.60.9 49.551.2% (theoretical yield = 55%)
substrate-to-inoculum ratio, 19 days
Methanogens I and II Methane Fed-batch anaerobic digestion: 37 C, Methane: 53.7% by methanogens I, 49.8% by II [19]
okara loading rate 1 g/l-day, 20 days
H2 -producing Hydrogen Batch fermentation: 60 C, pH 5.5, 4:1 Batchrate: 36 ml H2 /h, yield: 1.7 mol H2 /mol [93]
consortium okara-to-anaerobic digester sludge glucose equivalent
ratio
Continuous fermentation: 60 C, pH Continuousrate: 12 l H2 /l-day, yield: 2.3 mol
5.5, 4 h retention time, 5060% H2 as H2 /mol glucose equivalent
headspace gas
Clostridium Hydrogen Submerged fermentation: 35 C, pH Inhibition: by bacteriocins from lactic acid [94]
acetobutylicum 78 bacteria, can be reduced by heat treatment
IAM19012, 19013, at >50 C for 30 min
14194
Anaerobic mixed Hydrogen Continuous fermentation: 60 C, pH Rate: 19.9 l H2 /l-day, yield: 1.87 mol H2 /mol [95]
microflora 5.5, 4 h retention time hexose
B. subtilis RB14CS Iturin A Solid state fermentation: 79% Yield: 3.3 g/kg dry okara [99]
moisture, 20% (v/w) inoculum, 15 cm
substrate bed height
B. subtilis NB22 Iturin A Solid state fermentation: 25 C, 82% Yield: 2.0 g/kg dry okara with 15 g okara; [24]
moisture, 48 h dropped to 1.2 g/kg with 3 kg okara due to
temperature heterogeneity
Wolfiporia extensa Antioxidant Solid state fermentation: 24 C, 7.5 days Yield: 88.9 mg/g okara [104]
(Peck) Ginns polysaccharides
Flammulina velutipes Antioxidant Solid state fermentation: 74.5% Yield: 59.15 mg/g okara [125]
polysaccharides moisture, substrate C/N ratio 30.27
Candida albicans NRRL Antioxidant activities Solid state fermentation: 30 C, 50 g Improved quality. Radical scavenging activity: [17]
Y-12, C. guilliermondii and nutritional okara, 1 ml spore suspension as from 40% to 80%, antioxidant activity: from
NRRL Y-2075, improvement inoculum, 3 days 40% to 85%, chelating effects: from 55% to 80%,
Kluyveromyces total phenolic compounds: from 0.8 to 4.5 mg
marxianus NRRL gallic acid/g okara
Y-7571, Pichia pinus
& S. cerevisiae NRRL
Y-12632
Lentinus edodes Polyphenol Solid state fermentation: 12% inoculum Polyphenol: 22.9 mg gallic acid equivalent per [113]
(v/w), 77% moisture content, 24 days g okara
 A.A. Loman, L.-K. Ju / Process Biochemistry 51 (2016) 10461057
5. Specialty chemical production
5.1. Specialty chemicals from soybean meal
Recently, soybean carbohydrate has been considered for
production of a number of specialty chemicals by microbial fermen-
tation. Succinic acid is among the top twelve biomass-derivable
platform chemicals in a DOE study [76,77]. Thakker et al. [78]
studied succinate production using an engineered E. coli strain
HL27659k(pKK313)(pRU600). Soluble soybean meal carbohydrate
was acid hydrolyzed by 1% (v/v) sulfuric acid at 100 C for 1 h.
The hydrolysate was used as the fermentation substrate. The acid
hydrolysis did not completely monomerize the oligosaccharides,
mainly raffinose and sucrose. From an acid hydrolysate containing
431 mM total hexose sugars (22.61 mM raffinose, 5.68 mM sucrose,
139.62 mM galactose and fructose, and 75.19 mM glucose), 380 mM
total hexose sugars were consumed after 48-h fermentation and
312 mM succinate was produced. This corresponded to an 82%
molar yield of succinate from consumed carbohydrate. Without the
acid hydrolysis, sugar consumption was only 160 mM and the suc-
cinate production was 158 mM with a 98% molar yield of consumed
carbohydrate. The lower% molar yield (but much higher actual pro-
duction) from the acid-pretreated carbohydrate could be due to
inhibitory compounds generated by acid hydrolysis but concentra-
tions of these compounds were not reported. In another study, the
same authors studied fatty acid production from soluble soybean
meal carbohydrate using another engineered E. coli strain ML211
(MG1655 fadD fabR)/pXZ18Z [79]. Fatty acids are useful for produc-
tion of fuels, chemicals and healthcare products. Using this E. coli
strain with plasmid pXZ18Z carrying acyl carrier protein (ACP)
thioesterase and (3R)-hydroxyacyl-ACP dehydratase, 2.78 g/l fatty
acids were produced in shake flasks, with a yield of 0.21 g per g total
carbohydrate in medium.
Acetoin is a naturally occurring flavor compound present in
wine, honey, cocoa, butter, coffee and strawberry and an intermedi-
ate for the synthesis of diacetyl and 2,3-butanediol [80]. Consumer
demand increases the interest in commercial production of ace-
toin and production by fermentation is environmentally friendly
and more cost effective than the existing petroleum-based produc-
tion. Xiao et al. [81] produced acetoin using B. subtilis CICC 10025
fermentation and obtained higher yields from mixtures of sugar-
cane molasses and soybean meal hydrolysate than from a medium
based on pure sucrose, yeast extract and peptone. 35.4 g/l acetoin
was produced in a 5-l fermentor with the medium containing 22%
(v/v) molasses (125.5 g/l total sugars) and 27.8% (v/v) soybean meal
hydrolysate (38.9 g/l total sugars).
5.2. Specialty chemicals from soybean molasses
Sophorolipids are extracellular glycolipids with many potential
uses such as surfactants, emulsifiers, antimicrobial agents, and a
precursor to other specialty chemicals, e.g., sophorose and hydrox-
ylated fatty acids [82,83]. Soy molasses was used as a co-substrate
with oleic acid for the production of sophorolipids by Candida
bombicola [84]. Fed batch fermentation was started in a 4-l working
volume containing 333 g/l soy molasses (equivalent to 100 g/l sol-
uble carbohydrate) and 90 ml oleic acid. At 24 h and 48 h, another
200 g and 100 g equivalent soy molasses carbohydrate were added
respectively. At both 24 h and 48 h, 90 ml oleic acid was also added.
The sophorolipid yield was reported after isolation from fermenta-
tion broth. Only 21 g/l isolated sophorolipids were obtained, which
were significantly lower than the 79 g/l sophorolipids obtained by
using glucose as the co-substrate instead of soy molasses under
otherwise similar conditions. Two probable reasons were pro-
posed: oxygen transfer limitation due to high viscosity of soy
molasses, and incomplete utilization of stachyose and raffinose in
1053
soy molasses. In a subsequent study, the authors used a refined
product isolation procedure which would improve the above
reported yield of 21 g/l to 75 g/l in the system with soy molasses as
the co-substrate with oleic acid. Correspondingly, the sophorolipid
yield in the system with glucose as co-substrate with oleic acid
was increased from 79 to 100 g/l. They also investigated the use
of soy molasses not only as a co-substrate but also the sole nitro-
gen source, i.e., completely omitting yeast extract and urea from
the medium. In this case, the sophorolipid yield was 53 g/l, about
30% lower than the 75 g/l obtained in the system containing yeast
extract and urea. Nonetheless, considering the costs of yeast extract
and urea, the authors suggested that using soy molasses as the sole
nitrogen source would be economical [85].
Soybean molasses was also used for poly(hydroxyalkanoate)
(PHA) production. PHAs are produced by microorganisms as intra-
cellular granules. Being biodegradable polyesters, they have been
considered as green substitutes for petroleum-based polymers for
applications in drug-delivery, medicine, agriculture and other con-
sumer products. To reduce the production cost, use of inexpensive
feedstock is essential. Soy molasses was used to produce PHA by
Pseudomonas corrugata fermentation [86]. The study screened five
Pseudomonas strains for ability to metabolize sucrose, raffinose
and stachyose for PHA production. Unfortunately, none could use
stachyose or raffinose. Only P. corrugata could completely consume
sucrose. It grew to a 3.2-3.6 g/l cell dry weight (CDW) with 5% soy
molasses. Only 517% of CDW was crude PHA, significantly lower
than the percentages in cells grown on glucose or oleic acid (31%
and 61%). Full et al. [87] reported PHA production by strains isolated
for growth on raffinose, which was used as a model carbohydrate
of soybean molasses. They identified 3 Bacillus strains, among all
raffinose-metabolizing isolates, for higher PHA production. They
further selected 1 of the Bacillus strains for its ability to grow and
produce PHA in both aerobic and anaerobic conditions and to use
nitrate as an alternative electron acceptor (denitrification). Under
aerobic condition, this strain accumulated PHA at about 90% CDW
in media with raffinose, sucrose or one of the several other carbo-
hydrates tested as carbon source. (Mixed carbohydrates were not
tested.) They proposed that this strain can be used for PHA pro-
duction from carbohydrate or hydrolysate from soy molasses and
other soybean by-products.
Lactic acid is another reported fermentation product from soy-
bean based materials [88,89]. Soybean vinasse was generated by
yeast fermentation for ethanol production from soybean molasses.
During the yeast fermentation, only 47% of the carbohydrates were
used by S. cerevisiae. Lactic acid bacteria can metabolize the remain-
ing oligosacchairdes in soybean vinasse. The vinasse contained 35%
carbohydrates (22% raffinose, 11% stachyose and 2% galactose),
13.3% proteins, 27.8% lipids, 9.2% ashes, and 14.6% fibers. Karp et al.
[88] selected a Lactobacillus agilis strain (LPB 56) and produced
50 g/l lactic acid with higher than 80% yield from the vinasse. They
obtained similar yield in a pilot-scale process. Montelongo et al. [89]
optimized the conditions, pH 5.6 and 42 C, for lactic acid produc-
tion directly from soybean molasses using Lactobacillus salivarius.
They suggested additionally that supplementation of 0.5% yeast
extract to soy molasses reduced the fermentation time from 36 h
to 10 h and increased the production by 10%.
5.3. Specialty chemicals from soybean okara
5.3.1. Methane production
Methane production from okara was attempted using both
batch and fed batch fermentation. Muroyama et al. [19] studied
the important effects of okara feeding rate during the fed batch fer-
mentation for methane production by two methanogen cultures.
Maximum methane yield (53.7%) was achieved with the feeding
rate of 1.1 g/l-day for 20 days. On the other hand, the substrate-to-
1054 A.A. Loman, L.-K. Ju / Process Biochemistry 51 (2016) 10461057
inoculum ratio was highly important for batch fermentation [18].
The inoculum used in this study was prepared by incubating an
anaerobic digestion sludge at 36 C for 2 months with 0.5 g/l-day
okara feeding. Prior to use in the batch fermentation, the inocu-
lum was adjusted to have a moisture content of 93.5%. When the
inoculum was added at substrate-to-inoculum ratios (S/I) higher
than 0.9, methane production, per g volatile solids (a measure
of organic content of the substrate), decreased significantly from
496 ml methane at S/I of 0.930 ml at S/I of 1.6 and only 8 ml at S/I
of 3 [18]. Volatile fatty acids (VFAs), particularly acetic acid, are the
main source for methane production. Acetic acid alone contributed
to more than 73% of the methane produced [90]. However, at high
concentrations, they are also major inhibitors for methanogenesis;
for example, the inhibitory threshold was 17 mM for acetic acid and
33 mM for propionic acid [91]. Concentrations of formic, acetic and
propionic acids were measured in the study. None of these VFAs
were observed to accumulate in systems of S/I ratios of 0.50.9.
Formic acid did not accumulate at any S/I ratios of 0.52.0. At an S/I
ratio of 2.0, acetic acid concentration reached more than 120 mM
while propionic acid concentration was 30 mM (close to but still
lower than the inhibitory threshold of 33 mM). Therefore, the low
methane yield at higher S/I ratios could be explained by the inhi-
bition caused by accumulation of acetic acid beyond its inhibitory
threshold of 17 mM. Ammonia could also inhibit methane produc-
tion at concentrations higher than 4 g/l [92]; however, ammonia
did not reach inhibitory levels in the above study of methane pro-
duction from okara.
5.3.2. Hydrogen production
There have been several reports on hydrogen production from
soybean okara [9395]. These studies were aimed at improving
production rate and yield by batch or continuous fermentation pro-
cesses at different pH and temperature. Temperature was found
to be an important factor and hydrogen production rate and yield
generally favored thermophilic conditions [96] over the mesophilic
conditions [94]. Thermophilic flora showed higher degradability
of organic acids to hydrogen and could tolerate significantly more
ammonia inhibition than mesophilic flora [97].
Nokie et al. [94] investigated batch hydrogen production from
okara by C. acetobutylicum at 35 C. When okara was used without
pretreatment, production could not be maintained for longer than 5
days. This was found to be caused by the inhibitory effect of lactate-
producing bacteria. With okara pretreated at 5090 C for 30 min,
relatively stable production of 1.2 l H2 /l-day could be achieved,
although yield was still unsatisfactory at 0.52 mol H2 /mol hexose
[96]. Kim et al. [93] investigated the hydrogen production from soy-
bean okara in batch and continuous processes at 60 C and pH 5.5. In
batch fermentation they studied how addition of anaerobic digester
sludge to okara would affect production. They found that addition
of 20% (v/v) anaerobic digester sludge improved the production rate
to 36 ml H2 /h, from 11 ml H2 /h with only okara. They then evaluated
the production in continuous fermentation where hydraulic reten-
tion time (HRT) is an important factor. Maximum production rate of
12 l H2 /l-day was obtained at HRT of 4 h. They later coupled a mem-
brane filtration unit to the continuously stirred tank reactor (CSTR)
[95]. The content in CSTR was pumped to the filtration unit where
only permeate was removed and the un-degraded solid substrate
was circulated back to the CSTR. This configuration improved the
fermentation performance significantly: at 8-h HRT, the maximum
yield was 1.87 and 1.2 mol H2 /mol hexose equivalent, respectively,
for processes with and without the coupled filtration. Maximum
production rate of 19.86 l H2 /l-day was achieved at HRT of 4 h in
the filtration-coupled CSTR. Further decrease of HRT was found to
decrease the production rate drastically.
5.3.3. Antibiotic production
Iturin A, a lipopeptide antibiotic that suppresses the growth of
various plant pathogens [98], was reported to be produced by B.
subtilis solid state fermentation on soybean okara in several studies
[68,99101]. The iturin A production depended on initial pH, tem-
perature, carbon and nitrogen source supplement, and the aerobic
environment. In one study it was found that the iturin A produc-
tion was affected by the aerobic environment dictated by the bed
height of solid substrate [99]. This study was done with B. subtilis
in glass-column reactors with okara bed heights ranging from 5 to
20 cm. Maximum iturin A production, at 2.8 mg/g wet okara, was
obtained when the bed height was 15 cm. Previous research sug-
gested that lower aeration rate was favorable for higher iturin A
production [102]. It was thus proposed that the 15-cm bed height
was optimum because the oxygen supply was not too high and the
system was also not fully anaerobic. Slightly higher iturin A pro-
duction, 3.3 mg/g initial wet okara, was reported in another solid
state fermentation study [68]. The same group further optimized
the conditions and found that supplementation with glucose and
soybean meal could increase iturin A production significantly [100].
Under optimized conditions, pH 5.96.3 and 25 C, the iturin A yield
was 5.5 mg/g initial wet okara when the substrate (with moisture
content of 79%) contained 15 g okara, 1 g glucose and 1.8 g soybean
meal.
5.3.4. Improving antioxidant properties of polysaccharide
Polysaccharides in soybean okara are dietary fibers which
cannot be metabolized by the human body but often show phys-
iologically beneficial antioxidant activity [103]. The antioxidant
properties of okara polysaccharides can be improved by fermenta-
tion, mainly solid state fermentation using antioxidant-producing
microorganisms such as Wolfiporia extensa (peck) Ginns [104],
Flammulina velutipes [105], Bacillus natto [106] and B. subtilis B2
[107]. For solid state fermentation, operating conditions such as
initial moisture content, temperature, duration, and inoculum
size are important. For antioxidant production from okara, the
typical optimum ranges of these operating conditions are as fol-
lows: initial moisture content7080%, temperature2330 C
and duration1524 days. The antioxidant polysaccharide yield of
up to 88 mg per g dry okara has been reported [104].
In addition to the above specialty chemicals, soybean okara has
been studied for production of other bioproducts. For example, it
was used for production of citric acid by simultaneous saccharifi-
cation and solid-state fermentation using a cellulolytic Aspergillus
terreus and a citric acid-producing A. niger [108]. This process was
reported to produce 5.1 g citric acid per 100 g dry okara in 11
days. Other products made with okara as a fermentation substrate
include fibrinolytic enzymes [109], #-glucosidase inhibitor [110],
!-fructofuranosidase [111], surfactin [112], polyphenol [113], and
chitosan [16]. A summary of all these bioproducts obtainable by
okara fermentation is given in Table 3.
6. Summary
Although oil and protein are the principal valuable compo-
nents, soybean also contains a large amount of carbohydrate.
The carbohydrate mainly goes to the waste byproducts generated
during soybean processing. This review summarizes the recent
developments on utilization of these carbohydrate-rich soybean
processing byproducts for the production of biofuels, enzymes and
specialty chemicals by microbial fermentations (Fig. 2). For bio-
fuel production, hull and molasses are the preferred substrates
because they contain mainly carbohydrate and minimal protein.
With high protein content, soybean meal has a low C:N ratio for
being used as the major carbon-source substrate but can serve as
 A.A. Loman, L.-K. Ju / Process Biochemistry 51 (2016) 10461057
an effective nitrogen source for fermentation. Soybean meal has
also been used to induce production of lignocellulose-degrading
enzymes (e.g., xylanase, pectinase, polygalacturonase and cellu-
lase) by submerged or solid-state fermentation. Okara can be used
for production of biogas such as methane and hydrogen by anaero-
bic digestion. Some microorganisms have been used to improve the
antioxidant properties of okara polysaccharides and, thus, signifi-
cantly enhance its nutritional value. Many specialty chemicals, such
as organic acids, surfactants and antibiotics can be produced from
the soybean processing byproducts. Although research for utiliza-
tion of soybean carbohydrate has increased rapidly, the commercial
potential is yet to be realized. Further studies are of practical sig-
nificance, particularly for supporting development and scale-up of
the commercial production.
Acknowledgement
The funding support for this study was provided by research
grants from the United Soybean Board (Projects 1475, 2475, and
5256).
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