Rhizopus oryzae - DASGIP

Direct fermentation of
triticale starch to lactic acid
by Rhizopus oryzae
Zhizhuang Xiao, Meiqun Wu, Manon Beauchemin, Denis Groleau,
and Peter C.K. Lau*
Biotechnology Research Institute, National Research Council Canada,
Montreal, Quebec H4P 2R2, Canada
* Author for correspondence
Biotechnology Research Institute, National Research Council Canada,
6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada
Phone: +001 (514) 496-6325; Fax: +001 (514) 496-6265
Email: peter.lau@cnrc-nrc.gc.ca
Submitted: 1 December 2010; Revised: 19 February 2011;
Accepted: 10 March 2011
KEYWORDS: biorefinery; Rhizopus oryzae; triticale, starch platform, lactic
acid, calcium carbonate
ABBREVIATIONS: LA, lactic acid; DASGIP, Drescher Arnold
Schneider — Gesellschaft für Informations — und Prozesstechnologie; PLA,
polylactide, or polylactic acid
Abstract
The production of lactic acid from triticale starch was demonstrated
for the first time. Direct fermentation by Rhizopus oryzae in batch mode
led to a conversion yield of 0.74 g lactic acid/g starch. Kinetics analysis
of the fermentation suggested a multiphase process which consisted
of quick hydrolysis of starch, rapid glucose accumulation followed
by fast production of lactic acid. In batch fermentation mode, correct
p e e r r e V I e W
o r I g I N a l r e s e a r c h
timing and proper dosage of a neutralizing agent (calcium carbonate)
were found to be critical factors that affect the organic acid yield.
Addition of CaCO 3 at the time point when glucose accumulation
had reached its peak (24 h) resulted in the highest lactic acid yield.
The best ratio (by weight) of triticale starch to CaCO 3 for lactic acid
production was 1:1. It was important to maintain a pH of about 5
during the whole fermentation process. Fermentation carried out in
an optimized, commercially available small-scale parallel bioreactor
(DASGIP) yielded up to 0.87 g lactic acid/g triticale starch when
R. oryzae spores were directly added to the fermentation medium. Our
fermentation results indicated that triticale starch from this nonfood
crop is a promising renewable feedstock for production of lactic acid.
Introduction
Starch, being the most abundant storage carbohydrate on
earth, is a major feedstock for biorefineries outside of lignocellulosic
materials. 1 Starchy materials such as wheat,
corn, cassava, potato, rice, rye, and barley have been used
in lactic acid (LA; 2-hydroxypropionic acid) production, a valuable
carboxylic acid widely used in the food, cosmetic, pharmaceutical,
and chemical industries. 2,3 LA is best known for its utilization in
the production of the biodegradable, compostable, and biocompatible
polylactic acid (PLA), which provides an environment-friendly
alternative to biodegradable plastics derived from petrochemical
materials. 4–7 In the chemical industry, via decarboxylation, decarbonylation,
dehydration, or reduction with hydrogen, LA can be
converted to commodity chemicals such as acetaldehyde, acrylic acid,
and pro panoic acid. 8
Fermentative production of LA by microbes, in contrast to chemical
synthesis, e.g., by hydrolysis of lactonitrile using strong acids,
has several advantages, most importantly, the production of optically
pure L(+) or D(–)-lactic acid instead of a racemic mixture. 2–4,9,10 The
L(+)-isomer is the biologically important isomer and is a preferred
substrate in most industrial applications. Among the numerous
LA producers, the Lactobacillus, Bacillus, and Rhizopus genera are
considered as commercially viable, and each has its advantages
© mary ann liebert, inc. • Vol. 7 no. 2 • april 2011 INdustrIal BIotechNology 129

peer reVIeW
and disadvantages. 4 Unlike the lactobacilli, LA-producing Rhizopus
strains (R. oryzae, R. arrhizus) generate L(+)-LA as a sole isomer of
LA. 11,12 In addition, the Rhizopus fungi naturally produce the amylolytic
enzymes for starch saccharification; they require few complex
nutrients and only a small amount of inorganic salts for growth;
they are tolerant to low pHs; and fungal biomass is easier to separate
from the fermentation broth than bacterial biomass, thus facilitating
downstream processes. 12,13
As the first human-made hybrid crop of durum wheat (Triticum
spp.) and rye (Secale spp.), the century-old triticale (X Triticosecale
Wittmack) remains a largely unexplored crop, as its grain and forage
are used primarily as animal feed. 14–16 Triticale is generally known
for its adaptability to unfavorable soils, drought tolerance, cold
hardiness, disease resistance, and low-input requirements. Canadian
spring (e.g., Carman, Pronghorn, and AC Ultima) and winter varieties
(e.g., Pika and Bobcat) have superior adaptation to stress conditions
such as drought, marginal, or acidic soils. 14,16 Some 3 million acres of
triticale are projected to be grown in the Canadian Prairies by 2015
(Canadian Triticale Biorefinery Initiative; www.ctbi.ca). Like other
cereals, triticale is characterized by high starch and other carbohydrate
content. Total starch levels in triticale are equal to or higher
than for wheat. 16 Triticale grain dry matter contains 62.4%–70.9%
starch, a content that is dependent upon cultivar and year. 17 A recent
scanning electron microscopy study of triticale starch granules during
endosperm and seed development indicated unique and inherent
structures, such as channels or pores that may facilitate the flow of
hydrolytic enzymes into the granule matrix. 18 In addition, triticale
starch has the apparent advantages of low phenolic acids content
that otherwise negatively impacts starch hydrolysis, and a lower
gelatinization temperature (of 65–68°C) compared to those of barley,
wheat, and corn (in the range of 72–75°C; personal communication,
T. Vasanthan, University of Alberta).
Triticale starch is envisaged to provide an important carbohydrate
platform for a biorefinery value chain. In the present work, the feasibility
of LA production from triticale starch was assessed via fungal
fermentation by R. oryzae sb NRRL 29086 in a “direct,” or singlestep
mode, otherwise known as simultaneous saccharification and
fermentation (SSF; for reviews 3,9,12 ). We examined the effects of spore
inoculum size, temperature, and addition of calcium carbonate on the
production of LA in batch fermentations and compared the yields to
those obtained in a DASGIP parallel bioreactor system.
Material & methods
TRITICALE STARCH And AnALYSIS
Starch of 99% purity prepared from Triticale var. AC Ultima, a
Canadian winter cultivar, was kindly supplied by Dr. Thavaratnam
Vasanthan (University of Alberta) and Dr. Hong Qi (Alberta Agriculture
and Rural Development). The high-purity starch (

1×10 4 per mL. One gram of sterile calcium carbonate powder was
added to the culture medium at 0, 16, 20, 24, and 40 h during the
fermentation at 28°C. To determine the effect of CaCO 3 dosage on LA
production, different amounts of CaCO 3 were added to the medium
24 h after start of the fermentation. Fermentation without addition of
CaCO 3 was used as control. Each condition was carried out in duplicate.
Samples taken at different time points were subjected to analysis
for glucose and LA by HPLC and pH measurement.
FERMEnTATIOn In dASGIP PARALLEL BIOREACTOR
For initial scale-up of the fermentation, a 2 L DASGIP parallel bioreactor
system (DASGIP BioTools, Shrewsbury, Massachusetts, USA)
was used. R. oryzae spores were inoculated into 1 000 mL of Vogel
medium containing 3% (w/v) triticale starch at a final spore concentration
of 1×10 4 per mL. Initial pH of the fermentation medium was
adjusted to 5.0 ± 0.1 by addition of 28% NH 4 OH. The fermentation was
performed at 28°C. The dissolved oxygen was maintained by fixing
the agitation rate at 250 rpm and the inlet-gas flow rate at 0.1 vvm
(L/min). To avoid foaming, 200 µL of antifoam agent Mazu® DF 204
(BASF, Mount Olive, New Jersey, USA) was added to the medium.
Results
The effect of temperature on the conversion of triticale starch to
LA by the R. oryzae strain was first determined over four temperature
points; the highest yield of LA at two selected time points (24 h and
48 h) was obtained at 28°C (Figure 1). Inoculum size (spores/mL) also
affected the yield of LA, the highest obtained when the number of
R. oryzae spores was 1×10 4 /mL after inoculation of flasks containing
3% (w/v) triticale starch (Figure 2). The product yield decreased
by 23% when either a higher inoculum size (1×10 6 spores/mL) or a
much lower inoculum size (400 spores/mL) was used; however, good
yields were also obtained with an inoculum of 2 000 spores/mL and a
fermentation duration of 72 h.
Yield of lactic acid (g/L)
10
9
8
7
6
5
4
3
2
1
0
■ 24 h
■ 48 h
24 28 30 32
Temperature (°C)
Figure 1. Effect of temperature on the fermentation of triticale starch
into lactic acid by R. oryzae. Each data point represents mean ± SD
(n = 3). The p value of the data point for 28°C/48 h, compared to
others, is

peer reVIeW
A
Yield of lactic acid (g/L)
B
Glucose accumulation (g/L)
C
pH
25
20
15
10
5
no addition of CaCO3
1 g of CaCO3 at 0 h
1 g of CaCO3 at 16 h
■ 1 g of CaCO3 at 20 h
▲ 1 g of CaCO3 at 24 h
● 1 g of CaCO3 at 40 h
0 0 10 20 30 40 50 60 70
18
16
14
12
10
8
6
4
2
Fermentation time (h)
0
0 10 20 30 40 50 60 70
Fermentation time (h)
7
6
5
4
Figure 4. (A) Lactic acid yield as a function of CaCO3 addition at various
times; (B) glucose accumulation profile; (C) pH change during
the course of triticale starch fermentation to lactic acid by R. oryzae.
132 INdustrIal BIotechNology april 2011
no addition of CaCO3
1 g of CaCO3 at 0 h
1 g of CaCO3 at 16 h
■ 1 g of CaCO3 at 20 h
▲ 1 g of CaCO3 at 24 h
● 1 g of CaCO3 at 40 h
3
no addition of CaCO3
1 g of CaCO3 at 0 h
2 1 g of CaCO3 at 16 h
■ 1 g of CaCO3 at 20 h
1 ▲
●
1 g of CaCO3 at 24 h
1 g of CaCO3 at 40 h
0
0 10 20 30 40 50 60 70
Fermentation time (h)
(after 40 h), glucose consumption tapered off, while LA accumulation
reached a plateau.
When a larger inoculum size of 1×10 6 spores/mL was used,
observed LA production occurred earlier than in cases when smaller
inocula were used (data not shown); both glucose accumulation and
LA production were lower (this latter effect shown in Figure 1).
We examined next the influence of a time-course addition of
the neutralizing agent CaCO 3 on LA production. The results showed
that timing greatly influenced LA production (Figure 4A, Table 1),
glucose accumulation (Figure 4B), and pH values in the fermentation
medium (Figure 4C ). Compared to the control where no CaCO 3 was
added, CaCO 3 supplementation during the 20–40 h period (phases 3,
4, and 5) increased LA production; however, addition of CaCO 3 at
0 h and 16 h (phase 1 and 2) raised the pH to about 6.0 (Figure 4C ),
causing a delay as well as reduced glucose accumulation (Figure 4B )
and, hence, decreased LA production (Figure 4A, Table 1). CaCO 3
supplementation at 24 h yielded 74.0 ± 2.1 g LA from 100 g starch,
thus leading to an 80% increase in yield compared to when CaCO 3
was added immediately at the beginning of the fermentation.
The effect of CaCO 3 dosage (starch-to-CaCO 3 ratio) and quantity
of CaCO 3 on LA production was also examined (Table 2). The results
indicated that the best yield of LA was obtained at the starch-to-
CaCO 3 ratio (by weight) of 1:1. Adding more CaCO 3 provided no additional
benefit. However, a low CaCO 3 dosage (starch-to-CaCO 3 ratio of
6:1) was still beneficial since that fermentation produced 41% more
LA than without the neutralizing agent.
Instead of shake flasks, the DASGIP parallel bioreactor system was
employed to provide a comparison of LA yield. Initial experiments
examined the effect of temperature (30°C versus 28°C), rate of agitation,
inoculum concentration and format (spores versus mycelium),
aeration level, and influence of sparger design (Rushton-type impeller),
as well as the use of chemical antifoam (Mazu DF204). After several
initial runs, examples of promising yields of LA were determined
(Table 3). LA production reached 86.7±4.3 g from 100 g starch after
72 h of fermentation performed in a 2 L-scale DASGIP system. With
Table 1. Time effect of CaCO3 addition on the
production of lactic acid by R. oryzae
LA yield (g/L) LA yield (g/100 g starch)
Control (no CaCO3) 14.9±1.5 49.7±5.0
+ CaCO3 at 0 hr 12.3±0.1 41.0±0.3*
+ CaCO3 at 16 hr 12.8±0.1 42.7±0.3a + CaCO3 at 20 hr 19.1±0.3 63.7±1.0**
+ CaCO3 at 24 hr 22.2±0.7 74.0±2. 1 **
+ CaCO3 at 40 hr 20.3±0.1 67.7±0.3**
Note: Ratio of CaCO to starch = 1:3.
3
Each data point represents mean ± SD (n = 3).
anot significant; *, p value < 0.05; **, p value < 0.01; versus control

a fixed agitation rate of 250 rpm, aeration rates ranging between
0.03 and 0.1 L/min did not significantly affect the LA yield. However,
inoculum size was found to be important, as observed previously in
shake flasks experiments.
Discussion
This study is a benchmarking effort to assess the feasibility of LA
production from triticale starch via fungal fermentation employing
an R. oryzae strain, giving LA yields from starch (0.74–0.87 g/g)
that are competitive with other starchy materials. Yu and Hang 11
studied the direct fermentation of several starchy agricultural commodities
and found the following order of merit in terms of LA yield:
rice > corn > cassava > barley > oats. The yields of LA and mode
of production by various starchy materials are compared in Table 4.
Rice and corn yielded about 45 g and 35 g of LA from 100 g starch,
respectively, when Rhizopus spores were used as inoculum in the flask
experiments. 11,23 An oat starch conversion yield of 68 g LA/100 g
starch was achieved in 10 L airlift bioreactors when spores were
used as inoculum. 24 Sweet potato starch produced 72 g LA from 100
Table 2. Effect of CaCO 3 dosage on the production of lactic acid by R. oryzae
peer reVIeW
g starch in 5 L stirred bioreactors when freshly prepared R. oryzae
mycelium from spores was used. 25 Up to 85 g LA was produced from
100 g corn starch in 3 L airlift bioreactors when the starch was prehydrolyzed
by α-amylase, also using freshly prepared mycelium from
spores as inoculum. 26 In the DASGIP bioreactor system described
herein, our study shows that simultaneous saccharification and fermentation
of triticale starch by R. oryzae spores produced 86.7±4.3 g
LA from 100 g starting starch material — a yield that was about 10%
higher than those obtained in shake flasks.
LA fermentations by Rhizopus strains have been carried out at
temperatures in the range of 27°C to 35°C. 12 The optimal temperature
for LA production by R. oryzae strain NRRL 29086 was 28°C. Yu and
Hang 11 reported that increasing the inoculum size of R. oryzae strain
NRRL 395 from an initial level of 1×10 5 spores/mL to 3×10 5 spores/
mL or more did not improve the LA yield obtained with a number
of agricultural commodities including barley, oat, cassava, corn, and
rice. However, our results indicated that R. oryzae inoculum size
significantly influenced LA production, 1×10 4 spores/mL being the
optimal concentration.
CaCO3 dosage (g/ 100 mL) 0 0.5 1 2 3 6
Ratio of starch to CaCO3 - 6 3 1.5 1 0.5
Yield of lactic acid (g/L) 12.8±2.7 18.1±0.5 19.8±0.2 20.2±0.1 21.0±0.5 20.8±1.8
Yield of lactic acid (g/100 g starch) 42.7±9 60.3±1.7* 66.0±0.7* 67.3±0.3** 70.0±1.7** 69.3±6*
Note: Each data point represents mean ± SD (n = 3). The significance is calculated relative to the production of LA without CaCO3 (*, p value < 0.05; **, p value < 0.01).
Table 3. Lactic acid fermentation of triticale starch by R. oryzae using a 1L DASGIP parallel bioreactor
system
Inoculum size (spores/mL) Agitation speed (rpm) Aeration rate (L/min) Lactic acid yield (g from 100 g starch)
2.4 × 103 250 0.1 86.7 ± 4.3
1.0 × 104 250 0.1 82.3 ± 4.1
1.0 × 104 250 0.03 81.0 ± 4.0
Note: Each data point represents mean ± SD (n = 4).
Table 4. Comparison of results of the present work to previous reports
Carbon source/
feedstock
Inoculum type Fermentation vessel
Yield of lactic acid
(g/L)
Yield of lactic acid
(g/100 g starch)
Reference
triticale starch spore flask 22 74 this study
triticale starch spore DASGIP bioreactor 26.1 87 this study
rice spore flask 45 45 11
corn spore flask unknown 35 23
oat starch spore airlift bioreactor 51.7 68 24
sweet potato starch mycelium stirred bioreactor 43.3 72 25
corn starch mycelium airlift bioreactor 102 85 26
© mary ann liebert, inc. • Vol. 7 no. 2 • april 2011 INdustrIal BIotechNology 133

peer reVIeW
Little information is known about the kinetics of the directed
fermentation of starch to LA by R. oryzae. In our study, the profiles
obtained for starch, glucose accumulation, LA production, and pH
during the entire fermentation process suggested that LA production
proceeded according to the following: First, rapid hydrolysis of the
starch provided nutrients and energy for fungal growth and for the
amylolytic enzyme production (phase 1 and 2). Second, fast accumulation
of glucose was observed due to amylolytic enzymes secreted by
the fungus (phase 2 and 3). Third, rapid production occurred of LA
that was subsequently secreted into the fermentation medium (phase
4). The kinetics of LA production appeared similar when a much
higher inoculum size (1×10 6 spores/mL) was used compared to the
optimal inoculum size (1×10 4 spores/mL). However, the use of larger
inoculum sizes led to earlier production of LA, although yields were
lower. It is assumed here that cultures that were started with a larger
inoculum size grew faster and required more energy for growth than
those started with a smaller inoculum size. Hence, determination of
the appropriate inoculum size appears essential for optimal production
of LA by R. oryzae. As expected, pH dropped during the fermentation
as LA was being produced (Figure 3B ). Among several neutralizing
agents, including sodium hydroxide, ammoniacal solution, and
sodium bicarbonate, CaCO 3 was found to be most effective for LA
production from potato starch via R. oryzae. 25 Several other studies
involved the use of CaCO 3 , as well as experimenting on the time of
addition, dosage, and ratio of fermentation substrate-to-CaCO 3 . 11,23,26–29
Our results indicated that time of addition together with dosage of
CaCO 3 was an important factor for LA production. Addition of CaCO 3
immediately at the beginning of fermentation was not a good strategy,
as it actually reduced LA yields compared to the control conditions.
Most of the LA yield occurred between 24 and 40 h (phase 4 and 5).
Addition of CaCO 3 at the 24 h time point, at which time glucose accumulation
had reached its peak, resulted in the highest LA production
(Table 1). The best ratio (by weight) of triticale starch-to-CaCO 3 for LA
production was 1:1. With the addition of the right amount of CaCO 3 at
the right time, the pH of the fermentation broth could be maintained
around 5 during the whole fermentation process.
The DASGIP parallel bioreactor offers various features for automated
control while permitting accelerated process development. 30 In this
study, LA production at the 2 L scale under a set of optimized conditions
exceeded that observed in shake flasks. However, further challenges still
need to be met, such as higher LA yields (g/L) and a shorter fermentation
cycle. In this context, further optimization of key parameters, such as the
use, or not, of fresh mycelium needs to be considered. Also, to meet the
cost issues associated with starch processing, triticale flour and whole
grains should be considered in future experiments.
In conclusion, triticale offers an interesting prospect of a nonfood crop
for an important carbohydrate platform in a biorefinery value chain.
A C K n O w L E d G M E n T S
Funding of this work was provided by the Canadian Triticale
Biorefinery Initiative (CTBI) of the Agricultural Bioproducts
134 INdustrIal BIotechNology april 2011
Innovation Program of Agriculture and Agri-Food Canada. We thank
members of the CTBI for their continuous support, and John Lu and
Thava Vasanthan for discussion. We are grateful to S. Deschamp for
his expert help in chemical analysis. This manuscript is issued as
NRCC publication number 53354.
R E F E R E n C E S
1. Grull DR, Jetzinger F, Kozich M, Wastyn MM, and Wittenberger R. Industrial
starch platform — Status quo of production, modification and application. In:
Biorefineries — Industrial processes and products. Status quo and future directions.
Kamm B, Gruber PR, and Kamm M (eds), vol 2, 61-95, Wiley-VCH Verlag
GmbH & Co. KGaA, Weinheim (2006).
2. Wee YJ, Kim JN, and Ryu HW. Biotechnological production of lactic acid and its
recent applications. Food Tech Biotechnol 44, 163-172 (2006).
3. John RP, Anisha GS, Nampoothiri KM, and Pandey A. Direct lactic acid fermentation:
Focus on simultaneous saccharification and lactic acid production.
Biotechnol Adv 27, 145-152 (2009).
4. Gruber P, Henton DE, and Starr J. Polylactic acid from renewable resources.
In: Biorefineries — Industrial processes and products. Status quo and future
directions. Kamm B, Gruber PR, and Kamm M (eds), vol 2, 381-407, Wiley-VCH
Verlag GmbH & Co. KGaA, Weinheim (2006).
5. Datta R, and Henry M. Lactic acid: Fecent advances in products, processes, and
technologies — A review. J Chem Technol Biotechnol 81, 1119-1129 (2006).
6. Nampoothiri KM, Nair, NR, and John RP. An overview of the recent developments
in polylactide (PLA) research. Bioresource Technol 101, 8493-8501
(2010).
7. Pang X, Zhuang X, Tang Z, and Chen X. Polylactic acid (PLA): Research,
development and industrialization. Biotechnol J 5, 1125-1136 (2010).
8. Katryniok B, Paul S, and Dumeignil F. Highly efficient catalyst for the decarboxylation
of lactic acid to acetaldehyde. Green Chem 12, 1910-1913 (2010).
9. Reddy G, Altaf Md, Naveena BJ, Ventkateshwar M, and Kumar EV. Amylolytic
bacterial lactic acid fermentation — A review. Biotechnol Adv 26, 22-34 (2008).
10. John RP, Nampoothiri KM, and Pandey A. Fermentative production of lactic
acid from biomass: An overview on process developments and future perspectives.
Appl Microbiol Biotechnol 74, 524-534 (2007).
11. Yu RC and Hang YD. Kinetics of direct fermentation of agricultural commodities
to L(+) lactic acid by Rhizopus oryzae. Biotechnol Lett 11, 597-600 (1989).
12. Zhang ZY, Jin B, and Kelly JM. Production of lactic acid from renewable
materials by Rhizopus fungi. Biochem Eng J 35, 251-263 (2007).
13. Liu Y, Liao W, and Chen S. Co-production of lactic acid and chitin using a
pelletized filamentous fungus Rhizopus oryzae cultured on cull potatoes and
glucose. J Appl Microbiol 105, 1521-1528 (2008).
14. Salmon DF, Mergoum M, and Gomez-Macpherson H. Triticale production and
management. In: Triticale improvement and production. Mergoum M and
Gomez-Macpherson H. (eds), 27-37; Plant production and protection paper
179, Food and Agriculture Organization of the United Nations, Rome, Italy
(2004).
15. Oettler G. The fortune of a botanical curiosity — Triticale, past, present, and
future. J Agric Sci 143, 329-346 (2005).
16. Triticale Production and Utilization Manual. Alberta Agriculture, Food and Rural
Development (2005).
17. Buresova I, Sedlaclova I, Famera O, and Lipavsky J. Effect of growing conditions
on starch and protein content in triticale grain and amylose content in starch.
Plant Soil Environ 56, 99-104 (2010).
18. Li C-Y, Li W-H, Lee B, Laroche A, Cao L-P, and Lu Z-X. Morphological characterization
of triticale starch granules during endosperm development and seed
germination. Can J Plant Sci 91, 57-67 (2011).

19. Kandil A, Li J, Vasanthan V, Bressler, DC, and Tyler RT. Compositional changes
in whole grain flours as a result of solvent washing and their effect on starch
amylolysis. Food Res Int 44, 167-173 (2011).
20. Henriksson G, Akin DE, Hanlin RT, Rodriguez C, Archibald DD, Rigsby LL, and
Eriksson KL. Identification and retting efficiencies of fungi isolated from
dew-retted flax in the United States and Europe. Appl Environ Microbiol 63,
3950-3956 (1997).
21. Xiao Z, Wang S, Bergeron H, Zhang J, and Lau PCK. A flax-reting endopolygalacturonase-encoding
gene from Rhizopus oryzae. Antonie van Leeuwenhoek
94, 563-571 (2008) .
22. Smith CS, Salde SJ, Nordheim EV, Cascino JJ, Harris RF, and Andrews JH.
Sources of variability in the measurement of fungal spore yields. Appl Environ
Microbiol 54, 1430-1435 (1988) .
23. Hang YD. Direct fermentation of corn to L (+)-lactic acid by Rhizopus oryzae.
Biotechnol Lett 11, 299-300 (1989).
24. Koutinas AA, Malbranque F, Wang R, Campbell GM, and Webb C. Development
of an oat-based biorefinery for the production of L (+)-lactic acid by Rhizopus
oryzae and various value-added coproducts. J Agric Food Chem 55, 1755-1761
(2007).
25. Yen HW, Chen TJ, Pan WC, and Wu HJ. Effects of neutralizing agents on lactic
acid production by Rhizopus oryzae using sweet potato starch. World J
Microbiol Biotechnol 26, 437-441 (2010).
26. Yin P, Nishina N, Kosakai Y, Yahiro K, Park Y, and Okabe M. Enhanced production
of L(+)-lactic acid from corn starch in a culture of Rhizopus oryzae using
an air-lift bioreactor. J Ferment Bioeng 84, 249-253 (1997).
27. Zhou Y, Domínguez JM, Cao N, Du J, and Tsao GT. Optimization of L-lactic
acid production from glucose by Rhizopus oryzae ATCC 52311. Appl Biochem
Biotechnol 77-79, 401-407 (1999).
28. Liu Y, Liao W, Liu C, and Chen S. Optimization of L (+)-lactic acid production
using pelletized filamentous Rhizopus oryzae NRRL 395 Appl Biochem
Biotechnol 131, 844-853 (2006).
29. Huang LP, Jin B, and Lant P. Direct fermentation of potato starch wastewater
to lactic acid by Rhizopus oryzae and Rhizopus arrhizus. Bioproc Biosys Eng 27,
229-238 (2005).
30. Bareither R and Pollard D. A review of advanced small-scale parallel bioreactor
technology for accelerated process development: Current state and future
needs. Biotechnol Prog 27, 2-14 (2010)
peer reVIeW
© mary ann liebert, inc. • Vol. 7 no. 2 • april 2011 INdustrIal BIotechNology 135