Moriwaki‑Takano et al. Bioresour. Bioprocess.
https://doi.org/10.1186/s40643-021-00414-1(2021) 8:59

Open Access

RESEARCH

Production of spiculisporic acidby Talaromyces trachyspermus in fed‑batchbioreactor culture
Maki Moriwaki‑Takano1*, Chikako Asada2 and Yoshitosi Nakamura2

Abstract
Spiculisporic acid (SA) is a fatty acid-type biosurfactant with one lactone ring and two carboxyl groups. It has beenused in metal removers and cosmetics, because of its low propensity to cause irritation to the skin, its anti-bacterial
properties, and high surface activity. In the present study, we report an effective method for producing SA by select‑
ing a high-producing strain and investigating the effective medium components, conditions, and environments for
its culture. Among the 11 kinds of Talaromyces species, T. trachyspermus NBRC 32238 showed the highest productionof a crystalline substance, which was determined to be SA using NMR. The strain was able to produce SA under acidic
conditions from hexoses, pentoses, and disaccharides, with glucose and sucrose serving as the most appropriate
substrates. Investigation of nitrogen sources and trace metal ions revealed meat extract and F­ eCl3 as componentsthat promoted SA production. Upon comparing the two types of cultures with glucose in a baffle flask or aerationbioreactor, SA production was found to be slightly higher in the flask than in the reactor. In the bioreactor culture,sucrose was found to be an appropriate substrate for SA production, as compared to glucose, because with sucrose,
the lag time until the start of SA production was shortened. Finally, fed-batch culture with sucrose resulted in 60 g/L
of SA, with a total yield of 0.22 g SA/g sucrose and a productivity of 6.6 g/L/day.
Keywords: Biosurfactant, Spiculisporic acid, Talaromyces trachyspermus, Fed-batch culture, Bioreactor
Introduction
Surfactants are among the most versatile chemicals used
in a variety of industries, including detergents, paints,
paper products, pharmaceuticals, cosmetics, petroleum,
food, and water treatment (Elazzazy et al. 2015; Mahamallik and Pal 2017; Varjani and Upasani 2017). At present, domestic annual surfactant production in Japan ismore than 1.1 million tons, a number that is increasing
year by year (Year Book of Current Production Statics,
Chemical Industry 2019). Commercially available surfactants can be synthesized from petrochemicals, animal fats, plants, and microorganisms; however, most*Correspondence: mtakano@eng.u-toyama.ac.jp
1
Graduate School of Science and Engineering, University of Toyama, 3190
Gofuku, Toyama 930‑8555, Japan
Full list of author information is available at the end of the article

surfactant production relies on petrochemicals (Rufino
et al. 2014; Melo et al. 2015; De Almeida et al. 2016).
An increase in the amount of oil chemicals causes two
phenomena: an increase in environmental pollution,
which affects public health, and depletion of oil production within the next few decades (Frumkin et al. 2009).
Sustainable social growth demands the development of
new strategies to replace fossil fuel products with renewable and biodegradable materials. For the same reason,
surfactants made from natural resources using biotechnology, known as biosurfactants, are being focused on
to replace synthetics derived from petroleum. Biosurfactants produced by the metabolism of organisms arecharacterized by low toxicity, bioavailability, biodegradability, high effervescence, and environmental friendliness, and can be widely used in foods, pharmaceuticals,
cosmetics, etc. (Ahmadi-Ashtiani et al. 2020). These© The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material
in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material
is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds thepermitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​
mmons.​org/​licen​ses/​by/4.​0/.

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have been developed and used as multifunctional agents,
such as stabilizers, wetting agents, anti-bacterial agents,
humectants, emulsifiers, and anti-adhesion agents (Banat
et al. 2000; Vijayakumar and Saravanan 2015). However,
the mass production of many biosurfactants is difficultand expensive because of the low productivity of microorganisms and low availability of raw materials (Santos
et al. 2016).
These biosurfactants are biosynthesized primarily
as secondary metabolites and play an important role inmicrobial growth and localization. Based on the chemical
structure of the hydrophobic component, biosurfactants
are classified into four categories: (1) glycolipid-type, (2)fatty acid-type, (3) lipopeptide-type, and (4) polymertype (Raffa et al. 2015; Vijayakumar and Saravanan 2015).
Glycolipid-types include rhamnolipids, sophorolipids,
and mannosylerythritol lipids. For example, rhamnolipids are produced by Pseudomonas sp., and surfactin of
the cyclic lipopeptide is produced by Bacillus subtilis(Soberón-Chávez et al. 2005; Chen et al. 2015).
Spiculisporic acid (4,5-dicarboxy-4-pentadecanolide,SA) is a fatty acid-type biosurfactant (Ishigami et al.
2013). The tricarboxylic-type surfactant structure has
recently received attention as a commercially available
biosurfactant and a new biomaterial owing to its safety
for human health and the environment. It contains 2–3
carboxylic acids and the physiological properties of asurfactant. SA was discovered as a metabolite from Penicillium spiculisporum, which was isolated from the precipitate crystallized in the acidified culture broth of thefungus (Clutterbuc et al. 1931). Isotope uptake studies
and enzyme purification showed that SA biosynthesis
involves the condensation of lauroyl-CoA and 2-oxoglutarate (Måhlén 1971, 1973). The reaction is catalyzed bydecylhomocitrate synthase. Enantioselective synthesis of
SA has also been reported (Brandänge et al. 1984; Brown
et al. 2003). SA is used in commercial applications to prepare new emulsion-type organogels, superfine microcapsules (vesicles or liposomes), heavy metal sequestrants,
and in many other applications in specialty chemicals andbiotechnology (Hong et al. 1998; Pekdemir et al. 2000).
The raw material has low surface activity, which increases
in the derivatives (Yamazaki et al. 1983; Choi et al. 1993).
The derivatives promote transdermal absorption of the
moisturizer. Due to their physiological properties, thesehave potential use as new bioactive substances in thepharmaceutical, cosmetic, agricultural, and biotechnology industries.
Talaromyces trachyspermus is the perfect state teleomorph of P. spiculisporum. The genus Talaromyces was
first introduced by Benjamin in 1955 as teleomorphs of
the Penicillium species, with woven hyphae and soft cleistothecia ascomata (Benjamin 1955). It was redefinedPage 2 of 11

by Stolk and Samson in 1971 (Stolk and Samson 1971;
Fennell 1973). T. trachyspermus are characterized byconspicuously spindle-shaped, elliptical ascospores,
and a clear ascotic covering. T. trachyspermus has beenfound to produce a fatty acid surfactant, trachyspic acid[2-(carboxymethyl)-8-nonyl-9-oxo-1,6-dioxaspiro[4.4]non-7-ene-2,3-dicarboxylic acid], which inhibits heparanase (Shiozawa et al. 1995). Since the structure of the
product is similar to that of SA produced by P. spiculisporum, the strain was predicted to produce SA. Itsderivatives are found in T. trachyspermus and Aspergillus
candidus cultures (Wang et al. 2012, 2015; Kumla et al.
2014). While various reports have shown SA production
and characterization, its industrial production is limited.
It is manufactured in Japan by only a single source, Iwata
Chemical Industry Co. Ltd., which has established a manufacturing method for it. Annual production is estimatedto be 4–5 tons. Mass production and scale-up have inevitable problems in trying to achieve low production costs,
low environmental pollution, and advanced innovation in the industrial production of fermented products(Chakravarty et al. 2017). Fed-batch (semi-batch) operation is a practical cultivation technology to overcome thevarious problems that are faced when performing batchand continuous operation (El Moslamy 2019).
In this study, the effective production of SA by T. trachyspermus was investigated by changing the medium composition and culture conditions. In the shake flask-scale
step, the microbial response to medium components(carbon sources, nitrogen sources, and trace metals) andpH conditions was assessed for SA production. Scaledup cultivation was attempted in an aeration and stirringbioreactor, to achieve high SA productivity. The utility of
two types of culture was evaluated: normal batch culture
and fed-batch culture, in which two substrates of glucose
or sucrose solution were added to the culture medium.

Materials and methods
Microorganisms

Talaromyces trachyspermus NBRC 6440, 8890, 9861,
31360, 31757, 31907, 32238, 106931, and 106932, T.
assiutensis NBRC 30691, and T. assiutensis NBRC 31750
were purchased from the Biological Resource Centerof the National Institute of Technology and Evaluation(Japan). These fungi were maintained on potato dextrose
agar (PDA; Difco Laboratories, Detroit, MI, USA) slants
at 2–8 °C. A fungal pre-culture was prepared by cultivating the fungus on a PDA plate at 28 °C for 7 days.
Basic medium

Fungi were mainly cultivated in basic medium containing
the following (per L): 0.5 g ­KH2PO4, 0.5 g ­MgSO4·7H2O,
0.0915 g ­FeSO4·7H2O, 0.8 g ­(NH4)2SO4, 1.0 g corn steep

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liquor (CSL, Sigma-Aldrich, St. Louis, MO, USA) and100 g glucose. Glucose was mainly used as a carbon
source. All the reagents used, except CSL, were purchased from FUJIFILM Wako Pure Chemical Co. (Osaka,
Japan). The initial pH of the medium was adjusted to
2–4 using HCl aq. before sterilization. The medium was
autoclaved at 121 °C for 15 min. The carbon sources
used in this study were hexoses (glucose, galactose, andfructose), pentoses (xylose and arabinose), and disaccharides (sucrose and lactose) at an initial concentrationof 100 g/L. The nitrogen sources of the natural extracts
were casamino acid, casein (Sigma-Aldrich), malt extract(Difco Laboratories), meat extract (Kyokuto Pharmaceutical Industrial Co. Ltd., Tokyo, Japan), and Hipolypepton(Nihon Pharmaceutical Co. Ltd., Tokyo, Japan). The salts
used as nitrogen source were K­ NO3, ­NaNO3, ­NH4NO3,­(NH4)2SO4, and urea (FUJIFILM Wako Pure ChemicalCo.), which were added to the basic medium at a concentration of 5 g/L. The mineral components used were
­CaCl2, ­CoCl2, ­CuSO4, ­FeCl2, ­FeCl3, ­FeSO4, ­Fe2(SO4)3,­MnSO4, ­NiCl2, ­ZnCl2, and ­ZnSO4, which were added tothe basic medium at a concentration of 5 mg/L.
Batch flask culture

Cultivations using 13 types of filamentous fungi were
performed in an Erlenmeyer flask, as follows: hyphae
piece ~ 1 ­cm2 grown on the pre-culture PDA plate was
inoculated in a 500-mL Erlenmeyer flask with three baffles containing 100 mL of the medium. The flasks were
cultured with shaking on a rotary shaker at 28 °C and
140 rpm for 7 days.
After cultivation, the mycelium and SA were separated
from the culture solution using a filter paper ­(Advantec®
No. 131, 110 mm, ADVANTEC Toyo Kaisha, Ltd., Tokyo,
Japan). The filtrate was analyzed using HPLC. The solid
phase on the filter was washed several times with distilled water. After removing the water, ethanol was
poured above the filter paper to dissolve the SA, followedby collection of the filtrate. The solution containing SA
was evaporated at 50 °C for 24 h, and the weight of the
product was measured. After washing with ethanol, theresidue was dried in an oven at 90 °C for 24 h, to estimate
the growth of fungi in terms of dry cell weight (DCW).

Page 3 of 11

Fig. 1 Screening for an spiculisporic acid-producing fungus from
Talaromyces species. Cultivation was performed in a baffle flask for 7
days. The medium volume was 100 mL, containing 100 g/L glucose.
The culture conditions were pH 3.0 and a temperature of 28 °C

and 700 rpm, with an aeration rate of 0.5 vvm for 11–19
days.
Fed-batch cultures were performed in the same reactor with either glucose or sucrose. During the culture,
200 mL of 500 g/L sterilized sugar solution was addedfrom the insertion port of the bioreactor, at 6 and 11 days
for glucose, and at 4 and 8 days for sucrose.
HPLC assay

Culture broth samples filtered with filter paper were analyzed using HPLC (LC-10 system: Shimadzu Co., Kyoto,
Japan) using a refraction detector (RID-10A, Shimadzu
Co.) at 40 °C with 1.25 mM sulfuric acid as the mobile
phase, at a flow rate of 0.6 mL/min. The column used wasan IC Sep WA1 Wine Analysis Column (Transgenomic
Co., NE, USA).
NMR analysis

The refined product was analyzed using NMR, to identify the crystals secreted from T. trachyspermus. The
recrystallized SA was analyzed using 1H and 13C-NMR(400 MHz, JNM-ECS400, JEOL Ltd., Tokyo, Japan) after
evaporation of ethanol from the SA solution recoveredfrom the culture. The solvent used was 99.5% ethanol-d6,
and the inner standard was 0.005 v/v% tetramethylsilane.

Results and discussion
Bioreactor culture

Screening for a biosurfactant‑producing strain

An aeration stirring-type bioreactor with a volume of 2
L was used (Sakura Seiki Co., Nagano, Japan). Batch cultures were performed in 1 L of the medium with glucose
or sucrose in the bioreactor, which was autoclaved at
121 °C for 45 min. The cultivation was started with themycelium of T. trachyspermus inoculated into the bioreactor under aseptic conditions. It was controlled at 28 °C

Eleven strains of Talaromyces were compared for SA production, to select for the high-producing strains. These
were cultured with 100 g/L glucose in a baffle flask at pH
3.0, 28 °C for 7 days. The pre-experiment showed that thestrains produced SA under conditions of high substrateconcentration and low pH, under adequate aeration.
T. trachyspermus was able to produce SA, whereas T.

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assiutensis only grew (Fig. 1). SA dispersed into the culture medium in a crystalline form as soon as it was produced, because it is hardly soluble in water. The medium
became cloudy in the later stage of the culture. T. trachyspermus NBRC 32238 produced the highest SA yieldof 11.3 g/L, while the others produced a slight amountof SA, of no more than 3 g/L. The yield of NBRC 32238(based on sugar consumption) was 0.333 g SA/g glucose,
while the productivity was 0.012 g/L/day. The T. trachyspermus NBRC 32238 was selected as the most efficient
SA-producing strain and used in the subsequent studies.
Identification of the T. trachyspermus product using NMR

The substance secreted from T. trachyspermus NBRC
32238 was isolated as white needle-like solid withextremely high solubility in ethanol and poor solubility in water. A photograph of the SA crystal is shownin Fig. 2. Its molecular formula was determined by ESI
high-resolution mass spectrometry as C
­ 17H28O6 [m/z328.1880, calculated for 328.1886] having four degrees
of unsaturation. The 1H-NMR spectrum of the substancedisplayed signals characteristic for one methine [δH 3.01
(dd, J = 8.8, 2.8 Hz, H-5)], three methylenes [δH 2.63–2.43(m, ­H2-2 and H­ 2-3), 1.92–1.80 and 1.56–1.48 (each m,­H2-6)], and one methyl [δH 0.89 (t, J = 5.2 Hz, ­H3-15)] in
addition to other 16 aliphatic protons [δH 1.38–1.28 (m,H-7 ~ 14)] (Fig. 3a). These data indicate that compound
has one double bond and three carbonyls, which accountfor four degrees of unsaturation required by the molecular formula, so it must contain a ring. 13C- and DEPTNMR spectra exhibited signals corresponding to 17 carbons which can be classified as three carbonyl (δC 176.6,
173.6, and 172.3), one quaternary carbon (δC 86.5), one
methine (δC 50.7), eleven methylenes (δC 31.8–22.5), andone methyl (δC 13.4) (Fig. 3b). The partial connectivities

Fig. 2 A photograph of spiculisporic acid crystals. The product was
purified from T. trachyspermus NBRC 32238 using ethanol elution andrecrystallization

Page 4 of 11

of C2–C3 and C5–C6 were deduced from COSY and
HMQC correlations. These data of the substance werein good accordance with those from the commerciallyavailable spiculisporic acid produced by P. spiculisporum(Table 1). From the above reasons, we determined theisolated compound is SA (Fig. 4).
Effect of initial pH

The effect of initial pH on SA production was investigatedbecause the selected strain of T. trachyspermus NBRC
32238 produced the substance in a low pH medium. The
medium was prepared at an acidic pH of 2.0–4.0, and thestrain was cultured in the medium with 100 g/L glucose
at 28 °C for 7 days. Glucose consumption by the strain
increased with increasing pH up to a pH of 4.0 (Fig. 5).
When the initial pH was 3.0, a maximum SA amountof 11.2 g/L was produced, without linking the glucose
assimilation behavior. Low pH operation can avoid contamination problems in a scale-up culture during on-sitemanufacturing. The strain produced the crystalline form
SA at acidic pH. This phenomenon protects its own cellfrom the adverse effects of SA as a surfactant, because thesolubility of this substance increases with environmental
pH. Moreover, the salt changes the state of molecularaggregation in response to environmental pH: vesicles
are formed at pH in the range of approximately 5.8–6.2,
lipid particles at pH in the range of 6.3–6.6, and micelles
at pH 6.8 or higher (Yamazaki et al. 1983; Ishigami et al.
2000; Kunieda and Sakamoto 2010).
Substrate specificity

SA production by T. trachyspermus NBRC 32238 upon
using eight types of sugars (glucose, sucrose, galactose,arabinose, xylose, lactose, fructose, and maltose) as carbon sources was assessed. It was cultivated for 7 days inbasic medium containing 100 g/L of each sugar.
The strain assimilated and metabolized various carbohydrates (hexoses, pentoses, and disaccharides) (Fig. 6).
SA was produced from all the sugars at concentrationsin the range of 2.5–11.9 g/L. The highest SA yield of
11.9 g/L was produced upon use of glucose; the yield andproductivity were 0.14 g SA/g glucose and 1.7 g/L/day
based on sugar consumption, respectively. Sucrose was
the most consumed carbon source and had the largest
DCW, with similar SA production as the glucose culture.
The strain secretes acid-resistant invertase to decompose
sucrose into glucose and fructose.
Sugars, as energy sources, are involved in substanceconversion along metabolic pathways for cell growth.
Adequate substrates are essential for both fatty acid synthesis and promotion of the TCA cycle, since SA biosynthesis is a condensation reaction between lauroyl-CoA
and 2-oxoglutarate.

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Fig. 3 Analysis of the product from T. trachyspermus NBRC 32238 using NMR. Spectrum data of a 1H NNR b 13C NMR. The solvent used was 99.5%
ethanol-d6, and the inner standard was 0.005 v/v% tetramethylsilane

Selection of nitrogen sources

The basic medium culture containing ­(NH4)2SO4
and CSL resulted in a yield of at most 2.2 g/L of SA,
as described in the previous section. Nitrogen sources
affect the production of many materials as substrates
of enzymes and substances involved in the different
processes in microorganism cells. SA production was
evaluated upon using 10 alternate nitrogen sourcesfor ­(NH4)2SO4 in the medium. T. trachyspermus
NBRC 32238 was cultured for 7 days with these nitrogen sources (5 g/L) in a baffle flask containing 100 g/L
glucose. The strain grew and produced SA with eachnitrogen source (Fig. 7a). When inorganic sources wereused, glucose consumption was poor and SA production was approximately 0.5–15.0 g/L. Natural nitrogen sources improved glucose consumption and led torelatively high DCW and SA amounts. Meat extract
induced the highest SA production of 22.2 g/L. It isessential to determine which components of the meat
extract affect SA synthesis; however, this is challenging
since the components in the extract are private.
Different meat extract concentrations (0–5 g/L) were
investigated for SA production in basic medium with
100 g/L glucose at 28 °C for 7 days. SA production
increased with increasing meat extract up to 4.5 g/L
and reached a highest value of 23.3 g/L (Fig. 7b). The

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Table 1 NMR spectral data for commercial SA from P.
spiculisporum and T. trachyspermus’ product
PositionP. spiculisporum

T. trachyspermus

1

H-NMR spectral data
2

2.54

2.55

3

2.46

2.45

5

3.01 (dd)3.01(dd)6

1.87 (m), 1.48 (m)1.86 (m), 1.48 (m)7

1.28–1.39 (m)a1.28–1.38 (m)b8a1.28–1.39 (m)1.28–1.38 (m)b9a1.28–1.39 (m)1.28–1.38 (m)b10a1.28–1.39 (m)1.28–1.38 (m)b11a

1.28–1.39 (m)1.28–1.38 (m)b12

1.28–1.39 (m)a1.28–1.38 (m)b13a1.28–1.39 (m)1.28–1.38 (m)b14a1.28–1.39 (m)1.28–1.38 (m)b150.89 (t)0.89 (t)Fig. 5 Effect of initial pH on spiculisporic acid production by T.
trachyspermus NBRC 32238. The medium was adjusted to an initial
pH of 2.0–4.0, and the strain was cultured with 100 g/L glucose at
28 °C for 7 days. Error bars indicate standard deviations from three
independent experiments

13

C-NMR spectral data

1

176.6 (s)176.6 (s)2

27.6 (t)27.6 (t)329.4 (t)29.4 (t)486.5 (s)86.3 (s)4-COOH

173.6 (s)173.6 (s)550.7 (d)50.7 (d)5-COOH

172.3 (s)172.3 (s)627.8 (t)27.8 (t)727.7 (t)27.7 (t)829.4 (t)c29.4 (t)d929.2 (t)c29.2 (t)d10c29.2 (t)29.2 (t)d11c29.4 (t)29.4 (t)d12c29.5 (t)d1331.8 (t)31.8 (t)1422.5 (t)22.5 (t)15

13.4 (q)13.4 (q)a, bOverlapping signalsc, dInterchangeable signals

29.5 (t)Fig. 4 The structure of spiculisporic acid produced from T.
trachyspermus NBRC 32238

Fig. 6 Effect of carbon sources on spiculisporic acid production by
T. trachyspermus NBRC 32238. The initial substrate concentration
was 100 g/L. Cultivation was carried out at 28 °C for 7 days. Error bars
indicate standard deviations from three independent experiments

SA yield and productivity were 0.29 g SA/g glucose and3.3 g/L/day, respectively.
The amount of the target substance depends on thetype of nitrogen source to be added or the product lot.
This induces a large change in production, or, in somecases, no production of the target substance. Since
the meat extract used in this study was made from themeat of fishes and livestock, it contains several components that to be carbon sources and growth stimulatecompounds. Though these enhanced the growth andSA production of the strain, specific components that
affect the strain are unclear because details in the meat
extract are private. SA synthesis requires lipid production, the formation of 2-oxoglutarate in the TCA
cycle, and expression of enzymes involved in theseformations because it is produced by a condensation

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Fig. 7 Effect of nitrogen source on spiculisporic acid production
by T. trachyspermus NBRC 32238. a Comparison of spiculisporicacid production with various nitrogen sources. b Spiculisporicacid production with meat extract at concentrations of 0–5 g/L.
Cultivation was carried out with 100 g/L glucose at 28 °C for 7 day.
Error bars indicate standard deviations from three independent
experiments

reaction with lipids and organic acids. The formationof these enzymes necessitates the use of appropriate
nitrogen sources and the meat extract also affects thesereactions.
Effect of trace metal ions

The effects of metal ions involved in SA production were
examined to improve the production efficiency. Eleventypes of metal salts were added to the basal medium at
a concentration of 5 mg/L, and T. trachyspermus NBRC
32238 was cultured with 100 g/L glucose for 7 days.
The strain produced 5.2 g/L of SA in the control
case without the metal salts (Fig. 8a). Culture withthe addition of metal salts such as F
­ eSO4, ­Fe2(SO4)3,­FeCl3, ­MnSO4, and ­NiCl2 promoted SA production
to 2–3 times higher than that without addition. ­FeCl3
increased the glucose consumption to tenfold, as compared to the control culture, whereas the DCW was notdifferent (data not shown). This resulted in the induction of the highest concentration of SA (15.0 g/L).
The addition of iron-containing salt to the medium
significantly affected the glucose consumption and
SA production. The trivalent chloride iron, especially,
plays a positive role in the metabolism involved in SA

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Fig. 8 Effect of trace metal ions on spiculisporic acid production
by T. trachyspermus NBRC 32238. a Comparison of spiculisporic acidproduction with various metal compounds. b Spiculisporic acidproduction with F­ eCl3 at concentrations of 0–50 g/L. Cultivation was
carried out with 100 g/L glucose at 28 °C for 7 days. Error bars indicate
standard deviations from three independent experiments

production in T. trachyspermus because only ­FeCl3 promoted both glycolysis and SA production, while ­FeCl2,­FeSO4, and ­Fe2(SO4)3 did not.
The effects of addition of different concentrations of­FeCl3 to the medium (0–50 mg/L) were investigated.
Cultivation was performed under the same conditions
as described above. The produced SA increased withincreasing concentration of ­FeCl3, from 0 to 5 mg/L, andthen decreased upon further addition (Fig. 8b). The strain
was able to grow with 50 mg/L of ­FeCl3, but SA was notproduced at this concentration. The highest amount of
SA (18.6 g/L) was produced by the strain at 5 mg/L of­FeCl3.
Trace mineral components are required in the culture
medium for many physiological activities in the production of useful substances by microorganisms, for example, as an enzyme cofactor that acts on sugar metabolism,
substance production, substance synthesis, and cellretention. Iron ions play an important role in energy
acquisition by repeated oxidation and reduction on theelectron transport chain in the inner mitochondrial
membrane downstream of the TCA cycle. The trivalentchloride iron has a positive effect on the SA-producing
strain. ­FeCl3 enhances the expression level of related
enzymes in SA production, to increase the accumulationof fatty acids-CoA and 2-oxoglutarate generated in the
TCA cycle and downstream.

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Batch culture in flask or bioreactor

The selected media components were used in T. trachyspermus NBRC 32238 culture, to prove the effectivenessof SA production. This strain was cultivated in a flaskwith three baffles containing 100 g/L glucose at 28 °C and
140 rpm for 9 days. The nitrogen source and trace metalcompound used were 4.5 g/L meat extract and 5.0 g/L­FeCl3. Glucose consumption started from day 3 and was
almost depleted at day 7, and correspondingly, the cells
grew with the progress of culture and reached a DCW of
12.7 g/L at day 7 (Fig. 9a). The maximum amount of SA
was 29.0 g/L, which was 2.4-fold higher than that of thebasic medium culture. The SA yield of 29% on this strain
is close to that of 35% on the industrial strain of P. spiculisporum Lehman No.10-1 (Tabuchi et al. 1977).
In terms of pH behavior, SA production started
after the pH fell below 2.4, at day 3. The produced SA
decreased after a maximum amount, either because thestrain assimilated the product post-glucose decrease,

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or because it dissolved in the produced ethanol, to be
filtered through the filter paper. However, because theproduct is crystalline in low pH conditions, the reason
for this phenomenon is unclear. From the results of theflask batch culture, the growth of the mycelium cells
became constant after 4 days because of the decrease in
the pH of the medium. SA production stopped with glucose depletion on day 7. A scale-up of batch equipment
is necessary to make the process continuous and to produce the product stably and efficiently.
A bioreactor culture of T. trachyspermus NBRC 32238
was performed in a 1-L working volume with 100 g/L
glucose. The culture was carried out for 11 days at 28 °C
and 700 rpm with an aeration rate of 0.5 vvm. The strain
assimilated the substrate for 7 days and the DCW was
almost constant at approximately 11.2 g/L after 10 days
(Fig. 9b). SA production started on day 4 and reached avalue of 25.3 g/L at day 8, which was lower than that in
the flask culture. It also decreased after glucose depletion,
similar to that in the flask culture. In the glucose culture using both flask and bioreactor, the strain requires
3–4 days to decrease the pH to 2.4 and start producing
SA. Scale-up of the aerobic processes often reduces thebiomass yield and production of the target substance.
The use of filamentous fungi is particularly problematic
because of the entanglement of hyphae with the equipment. Dispersion is an important factor for substrates,
cells, and oxygen supply in bioprocesses using aerobic microorganisms. Optimization of these conditions
improves the production of the target substance.
SA production using fed‑batch culture

Fig. 9 Batch fermentation profiles for spiculisporic acid production
with different culture equipment. a Flask culture with three baffles
containing 100 mL medium with 100 g/L glucose at 28 °C for 9 days.
b Bioreactor culture by an aeration stirring-type reactor containing
1 L medium controlled at 28 °C and 700 rpm with an aeration rateof 0.5 vvm for 10 days. The cultivation was carried out with 100 g/L
glucose, 4.5 g/L meat extract, and 5.0 g/L ­FeCl3. Error bars indicate
standard deviations from three independent experiments

One of the major causes of decreased SA production
was substrate depletion in the batch culture. A fed-culture was performed to improve the SA production, withthe stepwise supply of an additional medium containing glucose to the bioreactor. The productivity of SA was
improved upon supplying sufficient oxygen and a carbon
source to the cells.
A fed-batch culture was performed with T. trachyspermus NBRC 32238 and glucose. Fermentation was firststarted with 100 g/L of substrate, 4.5 g/L meat extract,and 5.0 g/L ­FeCl3, under the same conditions as the batch
culture using the bioreactor. After incubation for 6 days,
and then subsequently, again at the interval of 6 days,
200 mL of 500 g/L glucose was added directly to the bioreactor at the point at which SA production stagnates.
The residual glucose concentration decreased to 19.5 g/L
and the cell grew to a DCW of 4.3 g/L in the early 6 daysof cultivation (Fig. 10a). At the same time, the produced
SA was only 0.66 g/L and hardly increased, although thestrain was growing by consuming glucose in the culture. After the addition of glucose, the sugar graduallyMoriwaki‑Takano et al. Bioresour. Bioprocess.

(2021) 8:59

Fig. 10 Fed-batch culture for spiculisporic acid production fromdifferent carbon source by T. trachyspermus NBRC 32238. a Glucose
culture, with feeding of additional sugar, at 6 and 12 days. b Sucrose
culture, with feeding of additional sugar, at 4 and 8 days. The cultures
were contained 4.5 g/L meat extract, and 5.0 g/L ­FeCl3 and operated
in an aeration stirring-type reactor containing 1 L medium controlled
at 28 °C and 700 rpm with an aeration rate of 0.5 vvm for 19 days incase of glucose culture and for 15 days in case of sucrose culture

decreased at the same rate as the first batch term, withincreasing DCW; in addition, SA production increased
drastically with growth and reached 39.5 g/L on day 11.
It further increased to 53.2 g/L on day 12, but still the
DCW hardly increased after glucose was added again,
because the mycelia amount in the reactor was limited.
The productivity of the fed-batch culture was 4.1 g/L/dayon day 12. It was constant at 47–55 g/L from day 12 to 19;
however, glucose was slowly consumed for maintenanceof the mycelia. The product reached its limit because of
the spatial inhibition in the reactor and other biochemical reasons of the strain, such as product inhibition andtoxicity. SA also did not decrease as in the batch culture
because adequate sugar remained around the myceliumin the fed-culture. The strain can consume sufficientsugar in this culture in the late term, while the depletionof glucose results in SA consumption in the batch culture.
Sucrose fed-batch culture was attempted using stepwise addition with T. trachyspermus NBRC 32238, as thestrain can produce SA from sucrose. The culture was performed using the same equipment and conditions, withthe replacement of glucose with sucrose at a concentration of 100 g/L. The substrate was fed on days 4 and 8,

Page 9 of 11which were the timings of growth and stagnation of SA
production, respectively. The residual sucrose decreased
to 58 g/L with the growth of the cells to a DCW of
12.6 g/L at day 4 (Fig. 10b). After the two additions, thegrowth of the mycelium remained almost constant fromday 4 until the end of the culture (15 days). The growthof the strain was promoted by sucrose, as evidenced by
the observation that the maximum growth was 5 days
faster than that with glucose. The SA yield was 12.1 g/L in
the sucrose culture at only day 4, although it was hardly
produced in the early term with glucose. Only 42 g/L ofsucrose consumption resulted in a DCW of 12.6 g/L andan SA amount of 12.1 g/L. After the first addition, sucrose
was rapidly consumed, from 179 g/L to 74 g/L, and simultaneously, SA amount increased to 44.7 g/L from day 4
to day 8 of the culture. Upon adding the sucrose solution
again on the 8th day, the SA yield reached 59.7 g/L andremained almost constant (56–60 g/L). Sucrose was consumed only to maintain the cells. The productivity of thesucrose fed-batch culture was 6.6 g/L/day on day 9, which
was 1.6-fold higher than that of the glucose culture. The
SA production was lower than that of P. spiculisporum
which produced 110 g/L from total glucose of 160 g/L by
4 times feeding for 11 days (Tabuchi et al. 1977). However, the rapid growth and SA production indicated thatsucrose is a better carbon source than glucose in fedbatch SA production. Using sucrose for the manufactureof a compound has the advantage that raw materials areeasily available, which keeps the unit cost relatively low.
A further increase in SA production is expected uponcarrying out continuous culture, which involves partiallyextracting the culture solution or sedimentation and separation of the product during the culture.
The fed-batch culture process is usually a step in an R
and D program that leads to large-scale industrial production by fermentation. The operation of a fed-batch
system for improving the productivity of a product isoften governed by a number of important engineering
considerations, such as improving the culture equipment and conditions. This process has the following
effects: (1) decreases the viscosity of the culture solution; (2) increases the density of the cells; (3) compensates for water loss; (4) extends the production time;(5) suppresses the Crabtree effect for high-concentration production; (6) suppresses catabolite repression
in secondary metabolite production; and (7) controls
the expression of genes with repressible promoters.
Fed-batch culture is desirable for a variety of industries
that deal with living organisms, such as the production of secondary metabolites, large-scale chemicals,
and pharmaceuticals that are produced by overexpressing recombinant or natural strains (Elsayed et al. 2015;
Krause et al. 2016).

Moriwaki‑Takano et al. Bioresour. Bioprocess.

(2021) 8:59

Conclusion
In the present study, we found that among 11 related
strains of Talaromyces, the novel ascomycetes T. trachyspermus NBRC 32238 had the highest production
capability for SA, a biosurfactant with multivalent carboxyl groups. The strain can produce SA effectively
from glucose and sucrose at low pH (no more than 3.0).
Low pH culture is an advantage that avoids contamination in bioprocesses using microorganisms. Nitrogensources and metals in the medium affect the production of secondary metabolites, including glycolytic
and metabolic pathways, redox reactions, and secretion of enzymes. The SA yield was improved upon in
the sequential identification of an optimum nitrogensource and trace metal component in the medium.
Meat extract and ­FeCl3 were found to be the appropriate components for this production. The factor ofa positive interaction becomes clear by examining
the detailed composition of the components and themetabolism when they are added. A typical problem
faced in the scale-up of fermentation processes is thateven though the cells continue to grow, the products
hardly appear. Reactor culture is different from thatcarried out in a flask, in terms of aeration, distribution, shear force, etc. The reactor culture of SA production reproduced the flask culture at the same level. In
this experimental system, the strain used the substrate
for simultaneous cell growth and SA production. To
improve the yield, fed-batch fermentation was applied
as a continuous production system. Periodic addition ofsubstrate increased the final SA amount and prevented
product consumption. Sucrose culture enhanced SA
production by T. trachyspermus. A remarkable SA of
60 g/L was achieved using the fed-batch process. The
fed-batch strategy is a promising, time-saving strategy
from a long-term perspective, because the process can
produce a high amount of substance in one tank. SA is
expected to be widely used in cosmetics, food, medical
materials, etc. In the near future, we hope that industrially produced SA using these strains is of application in
many fields.
Acknowledgements
Not applicable.
Authors’ contributions
Conceptualization, methodology, investigation, writing—original draft prepa‑
ration, MM-T; writing—review and editing, CA; supervision YN. All authors readand approved the final manuscript.
Funding
Not applicable.
Availability of data and materials
The dataset (graphs and tables) supporting the conclusions of this article isavailable.

Page 10 of 11

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
All authors agreed with this publication.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Graduate School of Science and Engineering, University of Toyama, 3190
Gofuku, Toyama 930‑8555, Japan. 2 Department of Bioscience and Bioindustry,
Tokushima University, 2‑1 Minamijosanjima‑cho, Tokushima 770‑8513, Japan.
Received: 7 April 2021 Accepted: 2 July 2021

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