Accepted Manuscript

Title: L-Lactic acid production from liquefied cassava starch

by thermotolerant Rhizopus microsporus: Characterization
and optimization

Authors: Srisakul Trakarnpaiboon, Nantana Srisuk, Kuakoon

Piyachomkwan, Shang-Tian Yang, Vichien Kitpreechavanich

PII: S1359-5113(17)30849-8
DOI: http://dx.doi.org/10.1016/j.procbio.2017.08.019
Reference: PRBI 11140

To appear in: Process Biochemistry

Received date: 21-5-2017

Revised date: 18-8-2017
Accepted date: 27-8-2017

Please cite this article as: Trakarnpaiboon Srisakul, Srisuk Nantana, Piyachomkwan
Kuakoon, Yang Shang-Tian, Kitpreechavanich Vichien.l-Lactic acid production from
liquefied cassava starch by thermotolerant Rhizopus microsporus: Characterization and
optimization.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2017.08.019

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L-Lactic acid production from liquefied cassava starch by

thermotolerant Rhizopus microsporus: Characterization and

optimization

Srisakul Trakarnpaiboon a, Nantana Srisuk a, Kuakoon Piyachomkwan b, Shang-Tian Yang c,

Vichien Kitpreechavanich a,*

a
Department of Microbiology, Faculty of Science, Kasetsart University, 50 Ngamwongwan Rd.,

Chatuchak, Bangkok 10900, Thailand

b
Cassava and Starch Technology Research Laboratory, National Center for Genetic

Engineering and Biotechnology, Pathum Thani 12120, Thailand

c
William G. Lowrie Department of Chemical and Biomolecular Engineering, Ohio State

University, 151 West Woodruff Avenue, Columbus, OH 43210, USA

* Corresponding author. Tel.: +66 2942 8389; fax: +66 2579 2081.

E-mail address: fsciwck@ku.ac.th (V. Kitpreechavanich).

Submitted to Process Biochemistry

1
Graphical abstract

Highlights

 A novel thermotolerant Rhizopus microsporus was isolated and characterized.

 L-lactic acid production from liquefied cassava starch at 40 oC was studied.

 84.3 g/L lactic acid at 0.84 g/g yield was obtained in batch fermentation at pH 5.5.

 Fed-batch gave 105–119 g/L of lactic acid with a yield of 0.93 g/g and productivity of

1.25 g/L/h.

 The strain can be used in SSF for lactic acid production from starchy substrates.

ABSTRACT

The thermotolerant Rhizopus microsporus DMKU 33 capable of producing L-lactic acid

from liquefied cassava starch was isolated and characterized for its phylogenetic relationship and

growth temperature and pH ranges. The concentrations of (NH4)2SO4, KH2PO4, MgSO4 and

ZnSO4·7H2O in the fermentation medium was optimized for lactic acid production from

2
liquefied cassava starch by Rhizopus microsporus DMKU 33 in shake-flasks at 40 oC. The

fermentation was then studied in a stirred-tank bioreactor with aeration at 0.75 vvm and agitation

at 200 rpm, achieving the highest lactic acid production of 84 g/L with a yield of 0.84 g/g at pH

5.5 in 3 days. Lactic acid production was further increased to 105–118 g/L with a yield of 0.93

g/g and productivity of 1.25 g/L/h in fed-batch fermentation. R. microsporus DMKU 33 is thus

advantageous to use in simultaneous saccharification and fermentation for L-lactic acid

production from low-cost starchy substrates.

Keywords: L-lactic acid; cassava starch; Rhizopus microsporus; simultaneous saccharification

and fermentation; thermotolerant

1. Introduction

An important industrial chemical, lactic acid is widely used in foods, cosmetics, and

pharmaceuticals [1-2]. Its use as a raw material for the production of poly(L-lactic acid) has also

become increasingly important in recent years [3]. Lactic acid production has been reported by

various microorganisms which exhibited various levels of product yield. For example, 0.93 g /g

of starch was found by Lactobacillus amylovorus NRRL B-4542 [4] whereas L. coryniformis

ATCC 25600 gave 0.89 g/g from cellulose [5]. Not only Lactic acid bacteria (LAB) but also

yeast and filamentous fungi showed capability on lactic acid production. Lactic acid production

from cellulosic sugar mixture by engineered Saccharomyces cerevisiae yielded 80 g/L with 0.66

g lactic acid/g sugar [6]. Rhizopus oryzae preferred starchy substrate for lactic acid fermentation

with the yield of 0.87 to 0.97 g/g of starch [7]. Among all of them, LAB are currently used in

3
industrial lactic acid fermentation for their high lactic acid productivity and yield from glucose

[1,8]. Although lactic acid also can be produced from less expensive starchy materials using

amylolytic LAB [9], the anaerobic bacterial fermentation has a fastidious nutrient requirement

and usually produces a mixture of D(–)- and L(+)-lactic acids that are difficult to separate for

generating polymer-grade lactic acid. Rhizopus species are important in biotechnology for their

ability to produce various extracellular enzymes [10] and organic acids [11]. Several species

including R. oryzae and R. arrhizus are known to produce high a amount of L-lactic acid from

various sugars and starch [12-16]. Compared to LAB, Rhizopus spp. have many advantages,

including the ability to use simple and low-cost media for growth and producing optically pure

L-lactic acid, which is easier and less expensive to recover and purify from the fermentation

broth [2]. In addition, the fungal biomass, which is rich in chitin, is easier to separate from the

broth and is a valuable byproduct [1]. Moreover, Rhizopus spp. have the ability to produce

amylolytic enzymes [17] and can use low-cost starchy materials as substrate in simultaneous

saccharification and fermentation (SSF) [8,18], which simplifies the production process and

decreases production cost.

However, one critical problem in SSF is the different temperature optima for enzymatic

hydrolysis of starch and the fermentation with mesophilic strains [8,11]. The starch hydrolysis

and saccharification are most efficient in the range of 40–70 oC, whereas mesophilic strains

usually do not grow well at temperatures above 35 oC. It is also desirable to operate SSF at a

higher temperature for increased starch solubility and lower broth viscosity, which could

significantly impact mass transfer and fermentation productivity. In addition, a higher

fermentation temperature of >35 oC can greatly reduce cooling requirement and cost, which is

also a major concern for aerobic fermentation, especially in the tropical region such as Thailand

4
[19]. Therefore, thermotolerant strains of Rhizopus can grow well at >40 oC are of high interest

for industrial L-lactic acid application [19].

The goal of this study was to screen for thermotolerant Rhizopus strains capable of

producing a high amount of L-lactic acid from cassava starch, which is cheap and readily

available throughout the year in Thailand. The best thermotolerant Rhizopus strain DMKU 33,

identified as R. microsporus, was characterized and studied for L-lactic acid production from α-

amylase-treated liquefied cassava starch in the optimized fermentation medium at 40 °C and pH

5.5. The SSF process was then scaled up and evaluated in a 5-L stirred-tank fermenter, which

showed high-titer and high-yield lactic acid production from liquefied cassava starch in batch

and fed-batch fermentations. This study demonstrated the potential of using thermotolerant

R. microsporus for industrial production of L-lactic acid from liquefied cassava starch as a low

cost substrate in Thailand.

2. Materials and methods

2.1. Microorganisms and cultivation

A total of 17 strains of Rhizopus spp. (DMKU 1, DMKU 2, DMKU 4, DMKU 5, DMKU

6, DMKU 7, DMKU 11, DMKU 16, DMKU 17, DMKU 18, DMKU 19, DMKU 20, DMKU 29,

DMKU 31, DMKU 33, DMKU 34 and DMKU 35), previously isolated from inoculums for

making a traditional Thai alcoholic beverage (loog pang lao) and sweetened glutinous rice (loog

pang kao mag) collected from different regions of Thailand, were used in this study [19]. All

isolates showed sporanigium and rhizoids as typical morphological characters to the genus of

Rhizopus. Unless otherwise noted, these cultures were grown on potato dextrose agar (PDA)

slants at 30 oC. Spores of these strains were collected by adding 5 mL 0.1% sterile Tween 80 to

7-day-old slant cultures and scratching the surface to obtain a spore suspension. Cells in the

5
suspension were counted with a haemocytometer and then adjusted to a desirable concentration

of 105–107 spores/mL. The stock spore suspension was stored in refrigerator at 4 oC and the

spore suspension was transferred to PDA slants every month to maintain viability.

2.2. Screening of thermotolerant Rhizopus spp. for L-lactic acid production at 40 oC

Primary screening of Rhizopus strains for high L-lactic acid production was conducted on

starch agar medium plates consisting of 3 g/L yeast extract, 10 g/L agar, 0.04 g/L bromocresol

green and 20 g/L cassava starch or liquefied cassava starch obtained by treating with -amylase

(Termamyl® Classic; Novozymes, Bagsværd, Denmark) at 95–100 °C for 2 h. A 5-mm-diameter

piece of the mycelial mat of each strain, grown for 5 days on a PDA plate, was obtained using a

sterile cork borer, then inoculated onto each starch agar medium plate and incubated at 40 oC.

The diameters of the growth and acid-forming areas, as determined by the color change of

bromocresol from green to yellow, were measured after 24 h incubation. Strains showing high

growth and acid formation were chosen for a second evaluation of L-lactic acid production in

liquid medium at 40 oC.

2.3. Identification of L-lactic acid-producing thermotolerant Rhizopus strains

PCR amplification and DNA sequencing of the ITS region of the selected isolates was

also performed according to White et al. [20]. The PCR reaction mixture (50 μL) contained: 1–

20 ng total DNA, 10 μM of each primer, 0.5 μL Taq DNA polymerase, 2.5 mM each of dATP,

dGTP, dCTP and dTTP, 2 mM MgCl2, and 10× PCR buffer in deionized water. The PCR

reaction was carried out in a thermal cycler (model TC9610; Labnet International, Edison, NJ)

under the following conditions: initial denaturation at 95 oC for 2 min, 30 cycles of denaturation

at 95 oC for 1 min, annealing at 55 oC for 1 min and extension at 72 oC for 2 min, and chain

elongation at 72 oC for 10 min. The sequences were compared pairwise by the BLASTN search

6
program [21], and assembled and aligned using MEGA7 (Molecular Evolutionary Genetics

Analysis version 7.0) [22]. A phylogenetic tree was constructed using the program provided

within the MEGA7 software package. Bootstrap analysis of the confidence levels of the clades

was carried out with 1000 replicates [23].

2.4. Selection of the highest L-lactic acid-producing thermotolerant Rhizopus strain

For secondary screening, the fermentation medium modified from Praneetratananon et al.

[24] was used, and consisted of 100 g/L liquefied cassava starch, 3.2 g/L (NH4)2SO4, 0.3 g/L

KH2PO4, 0.75 g/L MgSO4·7H2O and 0.04 g/L ZnSO4·7H2O. The medium was autoclaved at 121

°C for 15 min. Each 250 mL shake-flasks containing 50 mL of the sterile medium was inoculated

with 1 mL of a spore suspension (107 spores/mL) and incubated on a rotary shaker agitated at

200 rpm. Broth samples collected at 72 h were analyzed for lactic acid and the strain DMKU 33

with the highest lactic acid production was selected for further study.

2.5. Characterization of Rhizopus strain DMKU 33

The Rhizopus strain DMKU 33 was grown on malt agar using the slide culture method

[19] at 35 oC for 2 days to investigate its morphological characteristics. The length of the

sporangiophore, size of sporangium and sporangiospores, and rhizoid arrangement were

measured and photographed under a light microscope (Eclipse Ci-L; Nikon, Tokyo, Japan). The

growth pH was investigated between 1 and 7 in potato dextrose broth, and the growth

temperature was evaluated between 30 °C and 55 °C on PDA plates. The effect of temperature

on lactic acid production from liquefied cassava starch was investigated at 33, 35, 40, 43 and 45
o
C in 250 mL shake-flasks each containing 50 mL of the fermentation medium, inoculated with

107 spores, and incubated on a rotary shaker at 200 rpm.

7
 The ability of DMKU 33 to assimilate and produce lactic acid from various carbon

sources was also investigated in the optimized medium containing 5 g/L (NH4)2SO4, 0.6 g/L

KH2PO4, 0.3 g/L MgSO4·7H2O, 0.04 g/L ZnSO4·7H2O, and ~100 g/L of a carbon source. Each

flask containing 50 mL of the media was inoculated with 1 mL of spore suspension (107

spores/mL) and incubated at 40 oC for 3 days on a rotary shaker agitated at 200 rpm. During

fermentation, 2 g of sterile CaCO3 powder (2 g) was added to neutralize the lactic acid produced

in each flask at 24 h.

2.6. Optimization of mineral salts for L-lactic acid production from liquefied cassava starch

The concentrations of mineral salts in the fermentation medium were optimized for lactic

acid production from liquefied cassava starch at 40 oC in shake-flasks. The optimization was

done one factor at a time, in the following order: (NH4)2SO4, KH2PO4, MgSO4·7H2O, and

ZnSO4·7H2O. Each flask containing 50 mL of the media was inoculated with 1 mL of spore

suspension (107 spores/mL) and incubated at 40 oC for 3 days on a rotary shaker agitated at 200

rpm. To avoid dramatic pH drop due to lactic acid production, 2 g of sterile CaCO3 powder was

added to each flask at 24 h.

2.7. L-lactic acid production from liquefied cassava starch in a stirred-tank bioreactor

Lactic acid production from liquefied cassava starch by DMKU 33 was studied in a 5-L

stirred-tank bioreactor (Biostat B; Sartorius, Göttingen, Germany) containing 2.5 L of the

optimized medium at 40 oC with pH controlled by the addition of 10% (w/v) NaOH solution.

The bioreactor was aerated at 0.75 vvm and agitated at 200 rpm. Batch fermentation kinetics

were first studied with ~100 g/L liquefied cassava starch at pH 5, 5.5, 6, and 7, respectively, to

evaluate the pH effect. The effect of starch concentration on the fermentation was then studied at

pH 5.5 with an increased starch concentration of ~150 g/L, which was done by either adding all

8
starch in the beginning as in batch fermentation or in two times as in fed-batch fermentation.

Two fed-batch fermentations were carried out with different initial starch concentrations of ~100

g/L and ~130 g/L, respectively, in 2 L of medium, followed with the addition of 500 mL fresh

medium containing additional liquefied cassava starch, to bring the total amount of starch to the

initial concentration of ~150 g/L, at 36 h and 48 h, respectively, when the starch in the

fermentation broth was less than 70 g/L. The culture broth was taken every 12 h for analysis of

residual starch, glucose, lactic acid, and ethanol. After removing cell biomass by centrifugation

at 16,800 rpm for 5 min, samples were frozen-stored at -20 °C until analysis.

2.8. Analytical methods

Glucose, lactic acid, and ethanol present in samples were determined using a high

performance liquid chromatography (HPLC) system (Shimadzu Scientific Instruments,

Columbia, MD, USA) equipped with a refractive index detector (RID-10A) and a Bio-Rad HPX-

87H organic acid analysis column (Richmond, CA, USA) at 45 °C. The mobile phase was 0.005

M H2SO4 at 0.6 mL/min. The optical purity of lactic acid was determined by HPLC with a

spectrophotometric detector (Shimadzu SPD-20A, Japan) at wavelength of 230 nm. The

separation was performed on a Shodex ORpak CRX-853 column at 50 oC using 2 mM CuSO4 as

the mobile phase solution at flow rate of 1.0 ml/min.The concentration of starch (as the total

reducing sugar) was determined using the phenol-sulfuric acid method [25]. Dry weight of the

mycelium in the fermentation broth was determined by weighing the mycelial biomass after

washing with 6 M HCl and drying at 100 oC overnight. The lactic acid yield and productivity

were calculated based on the final concentration of lactic acid, total reducing sugar consumed,

and the fermentation time.

9
3. Results and discussion

3.1. Screening and identification of thermotolerant Rhizopus spp.

Among the 17 Rhizopus strains tested, 10 strains showed high growth and acid

production (based on the diameters of colony and yellow zone on agar plates) from liquefied

cassava starch at 40 oC (see Table S1). Among them, DMKU 33 produced the highest amount of

lactic acid in liquefied cassava starch medium at 40 oC (Table 1). DMKU 33 and three other

strains (DMKU 1, DMKU 5, and DMKU 17) were also able to grow on cassava starch. Based on

the ITS regions of the ribosomal operon, a phylogenetic tree for these 10 strains was constructed

and shown in Fig. 1. DMKU 33 was identified as R. microsporus while all the other 9 strains

were R. oryzae. Among them, only DMKU 34 was closely related to R. oryzae IFO4716

(AB097300), a lactic acid-producing strain, whereas the other 8 strains (DMKU 1, DMKU 5,

DMKU 6, DMKU 7, DMKU 17, DMKU 20, DMKU 29 and DMKU 35) were closely related to

R. oryzae CBS 406.51 (AB181330), a fumaric acid-producing strain. In general, R. oryzae can be

divided into two groups based on the primary organic acid produced when grown on glucose

[26]. One group produces primarily L-(+)-lactic acid, while fumaric acid and L-(+)-malic acid

are the main products of the other group. The ITS region sequence of ribosomal RNA-encoding

DNA could be used to classify Rhizopus oryzae in relation to organic acid production [27].

However, our results showed poor correlation between the ITS sequence classification and

fumaric acid production strains (see Table 1 and Fig. 1). R. microsporus DMKU 33, the highest

lactic acid-producing strain, was closely related to R. microsporus var. oligosporus (AY803941).

No genetic difference among various strains of R. microsporus could be detected using various

known DNA markers [28-30]. In this study, R. microsporus showed a great lactic acid-producer

than R. oryzae strains because the screening was done at 40 oC which was preference growth

10
temperature for R. microsporus [31] as can be seen from the biomass production (Table 1).

Therefore, R. microsporus produced higher lactic acid than R. oryzae at 40 oC. Kitpreechavanich

et al. [19] also reported that R. microsporus TISTR 3518 produced higher L-lactic acid from

starch at higher temperatures than R. oryzae TISTR 3523.

3.2 Characterization of R. microsporus DMKU 33

R. microsporus DMKU 33 was cultured on a PDA agar plate. The colony covered the

agar surface with a dense cottony growth which was initially white and then turned to grey

brown upon further incubation. The morphological characterization of DMKU 33 under a light

microscope showed rhizoid with an average length of 64.58 ± 4.23 μm. The average diameters of

sporangiophores, sporangia, and sporangiospores were 366.94 ± 8.35 μm, 110 ± 2.10 μm, and

4.17 ± 0.53 μm, respectively. The sporangiospores were smooth to slightly striated, and almost

round to slightly elongated. These morphological characteristics were consistent with other

members of the microsporus group [32-33].

R. microsporus DMKU 33 cultured in PDB exhibited growth at pH 2–7, but not at pH 1.

Clearly, DMKU 33 is an acid-tolerant strain with the ability to grow well at pH 5.5 or below,

which would favour the production of lactic acid in the undissociated form desirable for its

separation by solvent extraction [2]. DMKU 33 grew on PDA at temperatures ranging from 30

°C to 50 °C, but no growth was observed at 55 oC. This result was consistent with a previous

study showing that R. microsporus exhibited maximum growth at 45 °C [33]. Fig. 2 shows the

effect of temperature in the range of 33–45 oC on lactic acid production from 100 g/L liquefied

cassava starch in shake-flasks. Except for 45 oC, good lactic acid (46.3–56.1 g/L) and biomass

production (8.17–9.53 g/L) were obtained in 96 h with the maximum production at 40 oC (Fig.

11
2). A large amount of ethanol (10–15 g/L) was also produced in these fermentations. However,

low sugar utilization of less than 20 g/L and poor lactic acid production of only 4.1 g/L were

obtained at the higher temperature of 45 oC. It is noted that large amounts of well dispersed

mycelia were observed at 40 oC, while fewer mycelia at 43 and 45 oC and large clumps at 33 and

35 oC were present in these shake-flask fermentations. Therefore, 40 oC was determined as the

optimal temperature for lactic acid production from starch by R. microsporus DMKU 33.

The analysis of carbon assimilation is helpful for further characterization of Rhizopus

spp.[19]. Sixteen different carbon sources were tested for R. microsporus DMKU 33 and the

results are shown in Table 2. Glucose and fructose gave the best growth at 12.3 g/L and 14.5 g/L

of biomass in 48 h while lactose, raffinose and cellulose could not be utilized. As expected,

glucose and fructose also gave the highest lactic acid production of over 100 g/L in 48 h by R.

microsporus DMKU 33 cultured in shake-flasks. Among the low-cost starch substrates tested,

the liquefied cassava starch also gave a high lactic acid production of 81 g/L with 11.7 g/L of

biomass in 72 h. In addition, 100% optical purity of L-lactic acid was obtained from R.

microsporus DMKU 33 since only L–lactic acid was detected from the HPLC column (Shodex

ORpak CRX-853) which can be distinguish the D-and L-form [34]. R. microsporus DMKU 33

was thus selected for lactic acid production in a SSF process.

3.3. Optimization of mineral salts for lactic acid production from liquefied cassava starch

To optimize the concentrations of various mineral salts, including (NH4)2SO4, KH2PO4,

ZnSO4·7H2O and MgSO4·7H2O, in the fermentation medium, their effects on lactic acid

production from 100 g/L liquefied cassava starch as substrate were studied in shake-flasks and

the results are summarized in Table 3. (NH4)2SO4 was studied first as it provided the nitrogen

source required for spore germination, mycelial growth, and cell maintenance [35]. (NH4)2SO4

12
was also reported as the best nitrogen source for lactic acid production by Rhizopus sp. [36]. In

general, increasing its concentration up to 5 g/L also increased cell biomass and lactic acid

production. However, further increasing its concentration to 6 g/L did not enhance lactic acid

production, although cell biomass was further increased. In general, a lower C/N ratio gave

higher cell growth and fermentation rates [10], but lactic acid yield from sugar consumed would

be reduced when there was too much nitrogen source present in the medium [37].

KH2PO4 contains phosphate required for ATP generation, nucleic acid biosynthesis, and

fungal spore germination [38], and its effects on cell growth and lactic acid production were

studied in the medium with the optimized (NH4)2SO4 concentration of 5 g/L. As expected, poor

cell growth and little sugar utilization and lactic acid production were observed in the medium

without KH2PO4. The highest biomass (12.3 g/L) and lactic acid production (86.5 g/L) were

obtained at 0.6 g/L KH2PO4, which was consistent with a previous report that increasing the

KH2PO4 concentration from 0.1 to 0.6 g/L resulted in higher lactic acid production of 85 g/L (vs.

71 g/L) [39]. However, further increasing KH2PO4 to 0.9 g/L decreased both biomass and lactic

acid production.

The effects of ZnSO4·7H2O and MgSO4·7H2O were studied in media with optimized

(NH4)2SO4 and KH2PO4 concentrations (5 g/L and 0.6 g/L, respectively). The additions of Zn++

and Mg++ had minimal effect on cell growth but were beneficial to lactic acid production, with

the maximum lactic acid production obtained at 0.30 g/L MgSO4·7H2O and 0.04 g/L

ZnSO4·7H2O. Zhang et al. [36] reported that MgSO4 was required for lactic acid production by

Rhizopus. However, increasing MgSO4·7H2O in the range of 0.45–0.90 g/L had a negative effect

on lactic acid production (data not shown). The final optimized medium gave the highest lactic

acid production of 91.93 ± 1.51 g/L from 100 g/L liquefied cassava starch.

13
 The mineral salt contents also affected the morphology of the fungal mycelia grown in

shake-flasks. In general, well dispersed mycelia were observed in media containing appropriate

amounts of mineral salts. However, excessive amounts of (NH4)2SO4 (6 g/L), KH2PO4 (0.9 g/L),

and ZnSO4·7H2O (0.06 g/L) resulted in large mycelial clumps, which were unfavourable to lactic

acid production due to limited mass (oxygen) transfer [15].

3.4. Lactic acid production from liquefied cassava starch in a 5-L stirred-tank bioreactor

The SSF process for L-lactic acid production from α-amylase-treated liquefied cassava

starch by R. microsporus DMKU 33 was scaled up from shake-flasks to a 5-L stirred-tank

bioreactor. The process was optimized in pH and starch concentration, and the results are

discussed below.

3.4.1. Effects of pH

The effects of pH in the range of 5.0–7.0 were studied in batch fermentation with an

initial starch concentration of ~100 g/L. As can be seen in Fig. 3, there was little lactic acid

production in the first 24 h, which was the period when spore germination, cell growth, and

starch saccharification by secreted glucoamylase occurred. In general, good cell growth in the

form of small pellets of 1.44 ± 0.35 mm and good lactic acid production of 83.1–84.3 g/L at an

overall yield of 0.84 g/g total sugar consumed and productivity of 1.15–1.17 g/L/h were obtained

in fermentations at pH 5.0–6.0 (Table 4). Although pH in the range of 5.0–6.0 did not

significantly affect lactic acid production, ethanol production decreased from 16.5 g/L at pH 5.0

to 9.4 g/L at pH 6.0.

A significantly lower lactic acid production of 54.8 ± 0.66 g/L (yield: 0.78 g/g,

productivity: 0.76 g/L/h) along with some large fluffy mycelial clumps attached to the agitation

14
shaft and pH probe were observed at pH 7. The fluffy clump morphology caused problems in

mixing and hindered mass transfer in the stirred-tank bioreactor, resulting in low lactic acid

production [37]. The fermentation at pH 7 might also be limited by starch saccharification, as

indicated by the low glucose levels of 0.43 g/L at 12 h and 1.83 g/L at 24 h, probably because

glucoamylase had the optimal activity at pH 4.5–5.5 [40-41]. Moreover, the lactic acid

fermentation at pH 7 showed higher final biomass than those obtained from pH 5.0-6.0 (Table 4).

However, a pH below 5.0 would inhibit cell growth and the biochemical reactions involved in

lactic acid production [42]. Clearly, a pH between 5.0 and 6.0 would be most favourable for the

fermentation, which was consistent with the reported optimal pH range for R. oryzae and R.

arrhizus [7,43]. Therefore, further studies to optimize lactic acid production from liquefied

cassava starch were conducted at pH 5.5.

3.4.2. Effects of starch concentration

It is desirable to produce lactic acid at a higher concentration to facilitate its recovery in

downstream processing. A higher overall lactic acid yield could be obtained by extending the

batch fermentation with additional substrates since the initial substrate consumption was mostly

for cell growth with little lactic acid production while the lactic acid yield after the first 24 h was

much higher at ~0.93 g/g (vs. the overall yield of 0.84 g/g). Therefore, fermentation with a

higher amount of liquefied cassava starch (total concentration: ~150 g/L) was studied in batch

and fed-batch modes at pH 5.5 (Fig. 4). With the initial starch concentration of 153.4 g/L, 91.8

g/L lactic acid was produced in 72 h (Fig. 4A), with a productivity of 1.28 g/L/h and yield of

0.76 g/g (see Table 4), which was even lower than the yield of 0.84 g/g obtained in the

fermentation with ~100 g/L liquefied cassava starch. The lower lactic acid yield might be

because of the high initial substrate concentration, which could cause substrate inhibition as

15
reported for lactic acid bacteria [44]. Fed-batch fermentation is thus often used to alleviate

substrate inhibition by maintaining the substrate concentration below its inhibition level. Fed-

batch fermentation with an initial 102.7 g/L liquefied cassava starch and 41.4 g/L addition at 36

h produced 105.3 g/L lactic acid in 84 h and 118.8 g/L in 108 h (Fig. 4B), with an overall

productivity of 1.25 g/L/h and yield of 0.93 g/g. Fed-batch fermentation with an initial 131.3 g/L

liquefied cassava starch and the addition of 26.1 g/L at 48 h gave comparable lactic acid

production (104.2 g/L in 84 h and 116.2 g/L in 96 h, productivity: 1.25 g/L/h, yield: 0.90 g/g)

(Fig. 4C). Clearly, fed-batch fermentation gave a significantly higher lactic acid yield compared

to batch fermentation with the same total amount of substrate.

The higher initial starch concentration of ~150 g/L used in batch fermentation might have

reduced oxygen transfer and the dissolved oxygen (DO) level due to increased broth viscosity. A

low or sub-optimal DO level could reduce lactic acid production and yield as it has been reported

that a high DO level of 70–90% oxygen tension was needed for high lactic acid yield and

productivity [37]. Lactic acid productivity and yield were also reported to correlate with oxygen

transfer rate (OTR) and overall mass transfer coefficient kLa in an airlift bioreactor [45], in which

an initial starch concentration of 120 g/L gave the highest lactic acid production (yield: 0.87 g/g;

productivity: 2.51 g/L/h) by Rhizopus sp. MK-96-1196 in batch fermentation. Lactic acid yield

decreased 12% to 0.77 g/g and productivity decreased 21% to 1.98 g/L/h when the initial starch

concentration increased to 150 g/L, which caused a reduction in OTR due to reduced kLa.

However, in our study with ~150 g/L total substrate, both batch and fed-batch fermentations

gave a similar productivity of 1.24–1.28 g/L/h, which was higher than that (1.17 g/L/h) obtained

in batch fermentation with 113 g/L starch although final pellet biomass of batch and fed-batch

fermentations showed nearly concentration. (see Table 4)

16
3.5 Comparison to other studies

It has been reported that the thermotolerant R. microsporus TISTR 3518 produced more

glucoamylase with higher thermal stability than the mesophilic TISTR 3523 at 40 °C [19,46].

Peixoto et al. [47] reported a high level of thermostable amylase from thermotolerant R.

microsporus, which was advantageous to use in starch saccharification. Using the thermotolerant

DMKU 33 for lactic acid production from starch at 40 oC offered several advantages over the

mesophilic Rhizopus strains. Compared to 30 or 35 oC, SSF at 40 °C gave a higher efficiency in

starch saccharification, which could limit the fermentation and lactic acid production [19]. At the

higher temperature, the higher solubility of starch and lower solution viscosity also led to

increased conversion of substrate to lactic acid. R. microsporus with high amylase activities also

could save cost by eliminating the need to add glucoamylase or an amylolytic strain usually

required when an LAB is used in the SSF process [48].

Table 5 compares lactic acid production from starchy materials by various Rhizopus

strains. The thermotolerant R. microsporus DMKU 33 gave a high lactic acid yield of 0.93 g/g

liquefied cassava starch at 40 oC, which was higher than those reported for Rhizopus (0.67–0.90

g/g) and comparable to that usually obtained from glucose with lactic acid bacteria (0.85–0.95

g/g glucose) [8]. A high final lactic acid concentration of ~120 g/L was also obtained with

DMKU 33 in fed-batch fermentation. A much higher lactic acid production of 145 g/L in batch

fermentation and 231 g/L in fed-batch fermentation were obtained from glucose with R. oryzae

NRRL 395 immobilized in sponge-like cubes of polyurethane foam., in which calcium lactate

was produced and formed crystals (precipitates) that alleviated lactic acid inhibition and thus

increased lactic acid production [49]. It should be noted that cell immobilization to control

fungal morphology can also improve mixing, mass transfer, and thus the productivity of the

17
aerobic fungal fermentation, achieving a high productivity of 2.5 g/L/h [37]. The productivity of

1.25 g/L/h obtained with DMKU 33 cell pellets could be further improved by increasing cell

density through cell recycle or immobilization [1].

4. Conclusions

The thermotolerant R. microsporus DMKU 33 can produce L-lactic acid from α-amylase-

treated liquefied cassava starch at 40 oC with a high yield of 0.90–0.93 g/g starch, productivity of

1.25 g/L/h, and final titer of 105.3–118.8 g/L in a SSF process, offering a promising way for

lactic acid production. The fungal fermentation with the advantages of using low-cost starchy

substrates without addition of glucoamylase and producing optically pure L-lactic acid should be

able to alternative industrial fermentation process for lactic acid production.

Acknowledgments

This work was supported by the Thailand Research Fund through the Royal Golden

Jubilee PhD Program and Kasetsart University (grant no. PHD/0148/2552), Kasetsart University

Research and Development Institute (KURDI), the Center for Advanced Studies in Tropical

Natural Resources, National Research University–Kasetsart University (CASTNAR, NRU-KU),

and the Cassava and Starch Technology Research Unit (CSTRU). This work was performed

under the Core-to-Core Program supported by the Japan Society for the Promotion of Science

(JSPS), National Research Council of Thailand (NRCT), Vietnam Ministry of Science and

Technology (MOST), National University of Laos, Beuth University of Applied Sciences

(Berlin), and Brawijaya University (Malang, Indonesia).

18
19
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26
Figure Captions

Fig. 1. A phylogenetic tree of ITS sequences of Rhizopus strains selected in this study and

from the GenBank database, constructed by neighbour-joining using MEGA version 7.

Fig. 2. Effects of temperature on sugar utilization and lactic acid and ethanol production from

liquefied cassava starch by R. microsporus DMKU 33 in shake-flask fermentations. The

initial starch concentration was ~100 g/L and the fermentation time was 96 h. CaCO3 was

added to the concentration of 40 g/L at 24 h to buffer the medium pH.

Fig. 3. Effects of pH on lactic acid production from liquefied cassava starch by R.

microsporus DMKU 33 in stirred-tank bioreactor at 40 oC with pH controlled with 10%

NaOH solution. A: pH 5; B: pH 5.5; C: pH 6; D: pH 7.

Fig. 4. Effects of starch concentration on lactic acid fermentation by R. microsporus DMKU

33 in stirred-tank bioreactor at 40 oC. A. Batch fermentation with an initial starch

concentration of 153 g/L; B. Fed-batch fermentation with an initial starch concentration of

103 g/L and addition of 41 g/L at 36 h; C. Fed-batch fermentation with an initial starch

concentration of 131 g/L and addition of 26 g/L at 48 h.

27
Fig. 1

28
Fig. 2

29
Fig. 3

30
 160

140

120

Concentration (g/L)
100
Total Sugar
80 Glucose
60 Lactic acid
Ethanol
40

20

0
0 12 24 36 48 60 72 84
A Time (h)
120

100
Concentration (g/L)

80

Total Sugar
60
Glucose
Lactic acid
40
Ethanol

20

0
0 12 24 36 48 60 72 84 96
B Time (h)

140

120

100
Concentration (g/L)

80
Total Sugar
60 Glucose
Lactic acid
40
Ethanol

20

0
0 12 24 36 48 60 72 84 96
C Time (h)

Fig. 4

31
 Table 1. Rhizopus strains identified by ITS region sequencing and their ability to produce lactic

and fumaric acids from liquefied cassava starch in shake-flasks at 40 oC.

Lactic acid Fumaric acid Biomass

Strain Species Yield g/L Yield (g/L)

g/L
(g/g) (g/g)

DMKU 1* R. oryzae 2.63 ± 0.43 0.03 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 6.51 ± 0.27

DMKU 5* R. oryzae 11.93 ± 0.01 0.16 ± 0.00 1.76 ± 0.02 0.02 ± 0.00 6.26 ± 0.17

DMKU 6 R. oryzae 4.88 ± 0.06 0.07 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 5.52 ± 0.23

DMKU 7 R. oryzae 5.00 ± 0.03 0.07 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 10.1 ± 0.21

DMKU 17* R. oryzae 2.20 ± 0.02 0.03 ± 0.00 2.28 ± 0.03 0.03 ± 0.00 5.62 ± 0.20

DMKU 20 R. oryzae 6.49 ± 0.03 0.09 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 10.3 ± 0.42

DMKU 29 R. oryzae 8.69 ± 0.03 0.11 ± 0.00 4.26 ± 0.03 0.05 ± 0.00 6.26 ± 0.09

DMKU 34 R. oryzae 14.45 ± 0.43 0.15 ± 0.00 1.96 ± 0.03 0.02 ± 0.00 6.36 ± 0.45

DMKU 35 R. oryzae 6.90 ± 0.04 0.08 ± 0.00 6.45 ± 0.12 0.08 ± 0.00 7.96 ± 0.52

DMKU 33* R. microsporus 40.70 ± 0.12 0.63 ± 0.00 1.11 ± 0.01 0.02 ± 0.00 10.3 ± 0.64

*Strains also grew on non-liquefied cassava starch. Acids production in fermentation at 72 h was
determined by HPLC.

32
Table 2. Evaluation of various carbon sources for cell growth and lactic acid production by

R. microsporus DMKU 33 at 40 oC for 72 h.

Lactic acida Biomass

Carbon source
g/L Yield (g/g) (g/L)

D-Glucose 109.88 ±1.23* 0.93 ± 0.01 12.3 ± 0.24

D-Fructose 104.06 ±1.45* 0.89 ± 0.01 14.5 ± 0.59

D-Galactose 0.43 ± 0.01 0.09 ± 0.00 0.5 ± 0.04

D-Xylose 0.56 ± 0.01 0.09 ± 0.00 0.7± 0.02

Sucrose 45.1 ± 1.09 0.58 ± 0.01 10.9 ± 0.78

Lactose 0 0 0

Raffinose 0 0 0

Mannitol 2.48 ± 0.51 0.21 ± 0.04 5.1 ± 0.89

Sorbitol 31.42 ± 1.6 0.55 ± 0.02 7.1 ± 0.60

Maltodextrin 55.32 ± 0.98 0.81 ± 0.01 9.8 ± 0.34

Liquefied starch 81.04 ± 0.67 0.85 ± 0.01 11.7 ± 0.48

Soluble starch 43.88 ± 1.07 0.65 ± 0.02 9.7 ± 0.57

Cassava starch 42.49 ± 0.88 0.69 ± 0.02 8.8 ± 0.31

Corn starch 38.57 ± 0.67 0.67 ± 0.01 6.7 ± 0.93

Raw cassava starch 0.86 ± 0.01 0.43 ± 0.01 0.1 ± 0.78

Cellulose 0 0 0

*Maximum production at 48 h.

a. Lactic acid production was assayed in the optimized medium containing 40 g/L CaCO3 with an
initial pH of 6.0.

33
 Table 3. Effects of mineral salts on lactic acid production from liquefied cassava starch by
R. microsporus DMKU 33 in shake-flasks at 40 oC.

Concentration Residual Lactic acid Ethanol Biomass Cell

Mineral salt
(g/L) sugar (g/L) g/L Yield (g/g) (g/L) (g/L) morphologya

0 100.28 ± 1.41 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 1.79 ± 0.08 Little mycelia
2 70.23 ± 1.03 25.56 ± 1.06 0.85 ± 0.04 1.74 ± 0.32 3.68 ± 0.71 Small mycelia
3 53.98 ± 1.34 36.62 ± 1.19 0.79 ± 0.03 1.72 ± 0.41 5.98 ± 0.97
(NH4)2SO4 Well dispersed
4 44.21 ± 0.37 45.13 ± 1.47 0.80 ± 0.03 0.00 ± 0.00 8.06 ± 0.94
mycelia
5 43.70 ± 1.02 56.21 ± 1.44 0.99 ± 0.03 2.59 ± 1.18 9.89 ± 1.23
6 47.22 ± 1.02 55.24 ± 1.48 1.04 ± 0.03 5.81 ± 0.42 10.23 ± 0.21 Large clump
0 96.02 ± 0.10 1.61 ± 0.68 0.38 ± 0.06 0.00 ± 0.00 3.19 ± 0.53 Small mycelia
0.30 41.85 ± 0.92 56.41 ± 0.74 0.97 ± 0.01 7.20 ± 0.35 10.89 ± 1.49 Well dispersed
KH2PO4
0.60 18.61 ± 0.41 86.54 ± 1.05 1.06 ± 0.04 13.50 ± 0.64 12.32 ± 1.30 mycelia
0.90 9.21 ± 0.16 78.74 ± 0.65 0.86 ± 0.02 0.00 ± 0.00 10.75 ± 0.06 Large clump
0 12.64 ± 0.48 79.81 ± 1.16 0.91 ± 0.05 9.30 ± 0.14 11.18 ± 0.70
0.15 9.91 ± 0.41 84.14 ± 0.41 0.93 ± 0.01 11.20 ± 0.64 10.67 ± 0.20 Well dispersed
MgSO4∙7H2O
0.30 8.49 ± 0.20 89.39 ± 0.15 0.97 ± 0.00 9.20 ± 1.04 11.50 ± 0.41 mycelia
0.45 8.06 ± 0.16 81.40 ± 1.23 0.88 ± 0.03 10.30 ± 2.12 11.95 ± 0.04

ZnSO4∙7H2O 0 12.81 ± 0.27 79.76 ± 0.23 0.91 ± 0.00 4.90 ± 1.84 11.13 ± 0.06
Well dispersed
0.02 4.24 ± 0.16 83.28 ± 0.98 0.87 ± 0.04 11.07 ± 0.31 11.14 ± 0.08
mycelia
0.04 4.00 ± 0.28 91.93 ± 1.51 0.95 ± 0.05 9.67 ± 0.64 11.04 ± 0.11

0.06 6.40 ± 0.08 85.46 ± 0.95 0.91 ± 0.02 11.80 ± 1.11 11.83 ± 0.70 Large Clump

*Each shake-flask fermentation with 100 g/L liquefied cassava starch as substrate and 40 g/L CaCO3 for 72 h. All

conditions were studied in duplicate flasks.

a. 0.5-2.0 g/L of fugal biomass are little mycelia. 2.0-4.0 g/L of fugal biomass are small mycelia. And

more than 4 g/L are well mycelia.

34
Table 4. Lactic acid production from liquefied cassava starch by R. microsporus DMKU 33 in stirred-tank bioreactor at 40 oC and
various pH values.

Lactic acid Biomass Cell

Initial Starch Ferm Total sugar
Ethanol morphology
Mode pH starch added . time consumed (g/L)
Titer Yield Productivity (g/L)
(g/L) (g/L) (h) (g/L)
(g/L) (g/g) (g/L/h)

Batch 5 111.4 0 72 99.3 83.1 0.84 1.15 16.5 13.13 Pellet

5.5 113.3 0 72 100.4 84.3 0.84 1.17 11.0 12.39

6 107.8 0 72 98.3 82.5 0.84 1.15 9.4 12.95

7 106.4 0 72 70.3 54.8 0.78 0.76 4.6 17.61 Fluffy clump

5.5 153.4 0 72 117.5 91.8 0.76 1.28 7.2 12.71 Pellet

Fed- 5.5 102.7 41.4 84 113.7 105.3 0.93 1.25 3.0 12.97
batch Pellet
131.3 26.0 84 116.2 104.2 0.90 1.24 8.8 13.21

In fed-batch fermentation, the additional starch at the equivalent concentration in 2.5 L medium was added at 36 and 48 h,

respectively.

35
Table 5. Comparison of lactic acid production from starch by Rhizopus spp.

Strain Substrate Temperature Lactic acid Productivit Yield Reference

(oC) (g/L) y (g/L/h) (g/g)
R. oryzae NRRL 395 Corn starch 30 102 1.27 0.85 [35]
R. oryzae NRRL 395 Soluble corn 30 127 2.5 0.90 [37]
starch
R. oryzae NRRL 395 Hydrolyzed 30 16.7 0.29 0.24 [50]
cassava pulp
R. oryzae DMKU 12 Liquefied 35 77.7 1.2 0.67 [51]
cassava starch
R. oryzae RBU2-10 Hydrolyzed 30 88.7 1.84 0.75 [52]
rice starch
R. oryzae UV-40 Liquefied - ~115 ~2.6 ~0.85 [53]
sorghum
starch
R. arrhizus DAR 36017 Starch waste 30 19.5–44.3 0.49–1.11 0.85–0.96 [17]
R. arrhizus WEBL 0501 Waste potato 30 36.4 0.91 0.91 [36]
starch
R. arrhizus 36017 and R. Potato starch 30 15–20 0.40–0.98 0.85–0.92 [7]
oryzae 2062 wastewater
Rhizopus sp. MK-96- Corn starch 35 ~92 2.6 0.87 [45]
1196 mutant strain
Rhizopus sp. TISTR Cassava starch 40 14 0.19 0.75 [19]
3518
R. microsporus DMKU Liquefied 40 105.3–118.8 1.25 0.90–0.93 This study
33 cassava starch
Aspeergillus awamori & Soluble starch 30 120 1.6 0.76 [48]
Lactococcus lactis

36
R. oryzae NRRL 395 Glucose 37 145–231 1.42–1.83 0.93–0.95 [49]

37