Initial pH determines the morphological characteristics and secondary metabolite production in Aspergillus terreus and Streptomyces rimosus cocultures

Abstract

The influence of the initial pH on the morphology and secondary metabolite production in cocultures and axenic cultures of Aspergillus terreus and Streptomyces rimosus was investigated. The detected secondary metabolites (6 of bacterial and 4 of fungal origin) were not found in the cultures initiated at pH values less than or equal to 4.0. The highest mean levels of oxytetracycline were recorded in S. rimosus axenic culture at pH 5.0. Initiating the axenic culture at pH 5.9 led to visibly lower product levels, yet the presence of A. terreus reduced the negative effect of non-optimal pH and led to higher oxytetracycline titer than in the corresponding S. rimosus axenic culture. The cocultivation initiated at pH 5.0 or 5.9 triggered the formation of oxidized rimocidin. The products of A. terreus were absent in the cocultures. At pH 4.0, the striking morphological differences between the coculture and the axenic cultures were recorded.

Full text

ORIGINAL PAPER
Archives of Microbiology (2024) 206:452
https://doi.org/10.1007/s00203-024-04186-y
lamentous microorganisms, i.e., actinomycetes and la-
mentous fungi (Meyer et al. 2021; Brakhage 2013). The pH
level aects the solubility and transport of nutrients into
the cell, exerts stimulatory or inhibitory eects on the SM
biosynthetic pathways, and contributes to shaping the mor-
phological characteristics of fungal mycelium (Papagianni
2004). Notably, the optimal morphological form (dispersed
hyphae, clumps or pellets) and the recommended pH level
need to be determined experimentally for each microorgan-
ism and the target SM (Veiter et al. 2018). With regard to the
industrially important fungal genus Aspergillus, the forma-
tion of pellets is known to be initiated by the agglomeration
of spores, a process associated with the pH-dependent elec-
trostatic properties of spore surfaces (Wargenau et al. 2013).
As pointed out by Papagianni (2004), the tendency to form
pellets generally increases together with the increase of
medium pH. Carlsen et al. (1996) studied the pH-dependent
morphological development of Aspergillus oryzae myce-
lium and reported the formation of dispersed hyphae at pH
values between 3.0 and 3.5, the mixed (i.e., involving both
dispersed hyphae and pellets) morphology at pH between 4.0
and 5.0, and the strictly pelleted growth at pH values above
6.0. Furthermore, spore agglomerates were recorded at the
pH values higher than 4, while at lower pH levels (from
Introduction
The discovery and production of microbial secondary
metabolites (SMs) involve a great number of microbiologi-
cal methods that have been developed over nearly 100 years
since penicillin was discovered (Baral et al. 2018; Fleming
1929; Keller 2019). While the toolbox of experimental and
computational approaches continues to expand, the funda-
mental goals behind the biotechnology-oriented research on
SMs remain unchanged, namely to induce the biosynthesis
of potentially useful target metabolites, investigate their bio-
logical activity, and develop the bioprocesses which yield
the desired molecules at economically feasible productivi-
ties and titers (Nielsen and Nielsen 2017; Ramírez-Rendon
et al. 2022). The morphology and pH are among the factors
that strongly inuence the formation of SMs in the cells of
Communicated by Nischitha R.
Tomasz Boruta
tomasz.boruta@p.lodz.pl
1 Faculty of Process and Environmental Engineering,
Department of Bioprocess Engineering, Lodz University of
Technology, ul. Wólczańska 213, Łódź 93-005, Poland
Abstract
The inuence of the initial pH on the morphology and secondary metabolite production in cocultures and axenic cultures
of Aspergillus terreus and Streptomyces rimosus was investigated. The detected secondary metabolites (6 of bacterial
and 4 of fungal origin) were not found in the cultures initiated at pH values less than or equal to 4.0. The highest mean
levels of oxytetracycline were recorded in S. rimosus axenic culture at pH 5.0. Initiating the axenic culture at pH 5.9 led
to visibly lower product levels, yet the presence of A. terreus reduced the negative eect of non-optimal pH and led to
higher oxytetracycline titer than in the corresponding S. rimosus axenic culture. The cocultivation initiated at pH 5.0 or
5.9 triggered the formation of oxidized rimocidin. The products of A. terreus were absent in the cocultures. At pH 4.0,
the striking morphological dierences between the coculture and the axenic cultures were recorded.
Keywords Aspergillus terreus · Coculture · Secondary metabolites · Streptomyces rimosus
Received: 23 July 2024 / Revised: 10 October 2024 / Accepted: 26 October 2024 / Published online: 1 November 2024
© The Author(s) 2024
Initial pH determines the morphological characteristics and secondary
metabolite production in Aspergillus terreus and Streptomyces rimosus
cocultures
TomaszBoruta1· MartynaForyś1· WeronikaPawlikowska1· GrzegorzEnglart1· MarcinBizukojć1
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Archives of Microbiology (2024) 206:452
2.5 to 3.5) only the freely dispersed spores were observed
(Carlsen et al. 1996). In a study concerning Rhizopus sp.,
Nyman et al. (2013) demonstrated that the probability of
pellet formation at pH 5 was higher than at pH 4 or 6. Zhou
et al. (2000) noted the presence of Rhizopus oryzae pellets at
the pH values between 2.60 and 3.36, and also at 5.59. The
fungal species Mucor circinelloides was reported to display
pellet development due to the pH increase from the value
of 3 to 5.3 (Xia et al. 2011). In the case of actinomycetes,
it was shown for Streptomyces tendae that the decrease in
medium pH to the level of 4 leads to considerable increase
of pellet size (Vecht-Lifshitz et al. 1990). Notably, all the
aforementioned morphology-related transitions of lamen-
tous microorganisms take place at the pH values not exceed-
ing 6, i.e., under acidic conditions. Here, inspired by the
previously reported results, we conducted the cultivations of
a biotechnologically relevant lamentous fungal species of
remarkably rich SM catalogue (Amr et al. 2023; Boruta and
Bizukojć 2016; Guo and Wang 2014), namely Aspergillus
terreus (the producer of lovastatin, a cholesterol-lowering
drug), to describe its morphology and SM repertoire at pH
values between 2 and 6, i.e., within the pH range expected
to yield a diverse spectrum of morphological forms. For
comparative purposes, the cultures of a model actinomycete
(Pšeničnik et al. 2024) Streptomyces rimosus (the producer
of oxytetracycline, a broad-spectrum antibiotic) were inves-
tigated in parallel with A. terreus cultures. To the best of our
knowledge, the SM landscapes of A. terreus and S. rimosus
have never been investigated in relation to the pH-depen-
dent morphological diversity. In addition, the experimental
scope was expanded to include the two-species coculture
system involving A. terreus and S. rimosus, which was pre-
viously investigated in the context of secondary metabolite
production (Boruta et al. 2021, 2024) but without address-
ing the inuence of pH on the coculture outcomes. To date,
the eect of initial pH level of the medium on the reper-
toire of SMs in submerged cocultures of lamentous micro-
bial species has not been described in literature. It remains
unknown whether the pH-related observations made with
regard to axenic cultures could be used to predict the results
of the corresponding coculture variants as far as the bio-
synthesis of SMs and morphology are concerned. While the
conventional microbiological approaches relying on axenic
cultures (i.e., the cultures involving only a single organism)
are well-established, validated and relatively easy to con-
trol, the methods involving microbial cocultures are becom-
ing increasingly relevant in the SM-related studies (Nai and
Meyer 2018). Most importantly, they are eective in terms
of awakening silent biosynthetic gene clusters in microbial
strains through providing the conditions for stimulatory
interactions among the cocultured cells (Arora et al. 2020).
However, the bioprocess-related context is often overlooked
in the SM-oriented works involving microbial cocultures
and the focus is typically on the discovery of novel mol-
ecules rather than on the development and characteriza-
tion of cultivation processes. It should be emphasized that
the cocultures are associated with many process variables
absent from axenic cultures, i.e., the ones that reect the
interactions between two distinct microorganisms of dier-
ent nutritional and temperature preferences, growth rates,
biosynthetic repertoires, and morphological characteristics
(Diender et al. 2021). It has been demonstrated that the
inoculation ratio, medium composition, and relative inocu-
lation time contribute collectively to shaping the outcomes
of cocultures in terms of biomass growth and biosynthetic
capabilities (Goers et al. 2014; Kapoore et al. 2022). How-
ever, the pH-related aspects of SM production in lamen-
tous cocultures are yet to be addressed.
The aim of the study was to characterize the morphologi-
cal forms and the SM repertoire in submerged cocultures
and axenic cultures of A. terreus and S. rimosus under acidic
conditions.
Materials and methods
Microorganisms and sporulation conditions
The strains Aspergillus terreus ATCC 20542 and Strepto-
myces rimosus ATCC 10970 were used in the experimental
work. The spores of A. terreus were obtained via 10-day
cultivation on agar slants (malt extract, 20 g L− 1; casein
peptone, 5 g L− 1 ; agar, 20 g L− 1 ). For the sporulation of S.
rimosus, the bacterium was cultivated for 10 days on ISP
Medium 2 (BD, United States). The agar slants were stored
at 4 °C.
Cultivation medium
The liquid medium of the following composition was used:
glucose, 20 g L− 1; lactose, 20 g L− 1 ; yeast extract, 5 g L− 1;
KH2PO4, 1.51 g L− 1; MgSO4 ∙ 7 H2O, 0.52 g L− 1; ZnSO4 ∙
7 H2O, 1 mg L− 1 ; NaCl, 0.4 g L− 1; Fe(NO)3 ∙ 9 H2O, 2 mg
L− 1 ; biotin, 0.04 mg L− 1 ; trace element solution, 1 mL L− 1.
The composition of trace element solution was as follows:
H3BO3, 65 mg L− 1; CuSO4 ∙ 5 H2O, 250 mg L− 1 ; Na2MoO4
∙ 2 H2O, 50 mg L−1; MnSO4 ∙ 7 H2O, 43 mg L− 1 .
The pH of the medium was adjusted to the levels of 2.0,
3.0, 4.0, 5.0, and 6.0 (dependent on the variant) prior to
sterilization by using 1 M solutions of NaOH or HCl. After
15 min of autoclaving at 121 °C, the medium was cooled
down to the temperature of cultivation (i.e., 28 °C) and ali-
quots were taken to measure the post-sterilization pH val-
ues, which were equal to 2.0, 3.0, 4.0, 5.0, and 5.9. Hence,
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Archives of Microbiology (2024) 206:452
the values reported in this work as the initial pH, equal to
2.0, 3.0, 4.0, 5.0 and 5.9 are understood as the pH levels
of the cultivation media after sterilization. During the three
independent experiments performed over the course of the
study, the dierences in pH values between the correspond-
ing media variants after sterilization did not exceed ± 0.03.
Cultivation conditions
The inoculation of production medium was performed by
transferring spore suspensions of A. terreus and S. rimosus
(10 mL for each strain) to 200 mL of sterile liquid medium.
The spore suspension was prepared by removing the spores
from agar slants with the use of sterile plastic pipette and
suspending them in a portion of sterile medium. The number
of spores of each microorganism was adjusted with the use
of a hemocytometer to reach the nal spore concentration
in the production medium at the level of (1.0 ± 0.1) × 109
spores per liter. The cultivation was carried out in the labo-
ratory shaker Innova S44i (Eppendorf, Germany) at 28 °C
and 120 min− 1 . The cultures were propagated in 500 mL
at-bottom glass asks. The time of cultivation was equal
to 168 h.
Analysis of secondary metabolites
The biomass was removed via ltration and the liquid
samples were analyzed with the use of AQUITY-UPLC
apparatus coupled with SYNAPT G2 high-resolution mass
spectrometer (WATERS, USA). For chromatographic
(reversed-phase) separation of secondary metabolites, the
column BEH Shield RP18 (2.1 mm × 100 mm × 1.7 μm)
was used and the following water: acetonitrile gradient
was applied: 0 min, 100:0; 2.5 min, 80:20; 5.5 min, 70:30;
7.5 min, 60:40; and 14.0 min, 40:60. The eluents contained
formic acid at the concentration of 0.1% (v/v). The param-
eters of the mass spectrometry analysis were as follows:
sampling cone, 40 V; capillary voltage, 3 kV; extraction
cone, 4 V; temperature of the source, 120 °C; temperature
of desolvation, 200 °C; ionization: ESI−. For the putative
identication of secondary metabolites The Natural Prod-
uct Atlas (van Santen et al. 2022) was employed, under
the condition that the dierence between the observed and
calculated m/z values was less than 0.01. The identities of
mevinolinic acid, oxytetracycline, and butyrolactone I were
conrmed with the use of analytical standards. The standards
of mevinolinic acid and oxytetracycline were obtained from
Sigma-Aldrich (USA) and the standard of butyrolactone I
was purchased from Enzo Life Sciences (USA). The peak
areas corresponding to the assigned [M − H] − peaks were
determined by using TargetLynx software (WATERS, USA).
For mevinolinic acid and oxytetracycline, the concentration
values (in mg/L) were determined with the use of analyti-
cal standards. For the remaining metabolites, the peak area
values (in “counts”) were obtained based on the number of
counts recorded by the detector at a specied retention time.
The characterization of SM repertoire in A. terreus vs. S.
rimosus cocultures and the details regarding the experimen-
tal m/z values were described previously (Boruta et al. 2021,
2024).
Microscopic observations
The observations were carried out with the use of OLYM-
PUS BX53 light microscope, OLYMPUS DP27 camera,
and the software OLYMPUS cellSens Dimension Desk-
top 1.16 (Olympus Corporation, Tokyo). The microscopic
observations were performed by using two objectives. For
smaller objects, such as the pellets of S. rimosus, the 4×
objective was used (calibration X: 1.15 μm/pixel, calibra-
tion Y: 1.15 μm/pixel). Whenever the object was too large
to be observed via the 4× objective, the 2× objective was
employed (calibration X: 2.3 μm/pixel; calibration Y:
2.3 μm/pixel). Each microscopic image was presented with
a scale bar.
Statistical analysis
Three independent experiments were performed (n = 3).
Each result was presented as mean value ± standard devi-
ation. The two-sample t test with the signicance level
α = 0.05 was applied to determine whether the dierences
between the variants were statistically signicant. All cal-
culations were carried out with the use of OriginPro 2017
software (OriginLab Corporation, USA).
Results
The shake ask cocultures and axenic cultures involving A.
terreus and S. rimosus were initiated at 5 dierent pH lev-
els (2.0, 3.0, 4.0, 5.0, and 5.9). The investigation of experi-
mental variants encompassed the microscopic observations
of lamentous morphology, the analysis of SM repertoire
based on ultra-performance liquid chromatography coupled
with mass spectrometry (UPLC-MS), and the measurement
of nal pH levels of the cultures.
Morphology
Filamentous microorganisms in the submerged cultures
evolve in the following hyphal micromorphological (i.e.,
to be observed with the use of microscope) structures.
These are unbranched hyphae, branched hyphae, clumps
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Archives of Microbiology (2024) 206:452
a morphological peculiarity was recorded, manifested by
the development of irregular, large, and structurally diverse
biomass agglomerates of various shapes (Fig. 2a), which
were too large to be observed by means of a microscope
and thus classied as macromorphological forms. Similar
morphological behavior was noted in the case of S. rimosus
axenic variants again (Fig. 2c). However, the most inter-
esting observation was made with regard to the coculture,
which diered greatly from the axenic counterparts initiated
at pH 4.0. Specically, the co-cultivation of A. terreus and
S. rimosus resulted in the formation of fungal-like pellets
with dense cores and rather smooth, probably shaved, outer
regions (Fig. 1c). Hence, the coculture which was started at
pH 4.0 displayed the pelleted growth that could be moni-
tored microscopically, as opposed to the corresponding
axenic cultures of A. terreus and S. rimosus.
The pellets that were formed in the axenic cultures of A.
terreus at pH 5.0 were similar to the ones observed at pH
3.0, albeit with less hairy outer regions (Fig. 1a). In the case
of S. rimosus, the pH value of 5.0 was the lowest for which
the pelleted growth occurred, however, the resulting pellets
varied greatly in terms of shape, being rather irregular than
spherical (Fig. 1b). Importantly, both the A. terreus-like and
S. rimosus-like lamentous structures were clearly visible
in the coculture at pH 5.0. One could notice the presence
of larger pellets (resembling the pellets of A. terreus), the
smaller pellets of various shapes (most probably represent-
ing S. rimosus), and the clumps and dispersed biomass of
unknown microbial origin in the pH 5.0 coculture (Fig. 1c).
It was noted that the pellets of A. terreus (Fig. 1a) and S.
rimosus (Fig. 1b) at pH 5.9 were larger than the ones found
in the remaining axenic variants. Regarding the coculture
at pH 5.9, solely the S. rimosus-like pellets were visible in
the cultivation broth (Fig. 1c). Among the tested cocultures,
only the variant propagated at pH 5.9 lacked any fungal-like
lamentous structures.
To sum up, the cocultures initiated at pH 2.0, 3.0, and
4.0 revealed the presence of A. terreus-like pellets and no
traces of morphological forms resembling S. rimosus. The
opposite scenario (i.e., absence of A. terreus-like structures
and the presence of S. rimosus-like forms) was recorded at
pH 5.9. Finally, the microscopic images indicated that the
coculture initiated at pH 5.0 promoted the simultaneous
growth of A. terreus and S. rimosus.
Production of secondary metabolites
The UPLC-MS analysis of cocultures revealed the pres-
ence of 6 SMs originating from S. rimosus and previously
reported for the A. terreus vs. S. rimosus system (Boruta
et al. 2021, 2024). This group of molecules included oxy-
tetracycline, an antibiotic identied and assayed by using
and pellets. Clumps are formed from entangled hyphae but
have no distinctive core. Their shape is irregular. Pellets are
spherical hyphal structures with the distinctive core in their
center (Metz and Kossen 1977; Bizukojć and Ledakowicz
2010).
The morphological dierences recorded among the
investigated variants were striking (Figs. 1 and 2). At pH
2.0, A. terreus propagated in the form of moderately dense,
hairy clumps with irregular boundaries formed by the radi-
ally growing laments (Fig. 1a).These clumps fell apart
from the spherical shape and lacked distinctive cores.
Importantly, in this case one did not observe any formation
of pellets with distinct cores that are normally formed due
to spore agglomeration in Aspergilli, including Aspergillus
terreus (Bizukojć and Ledakowicz 2010). Still, the loose
branched and unbranched hyphae typically found at low
pH values (Papagianni 2004) were not visible here. Mark-
edly dierent behavior at the initial pH 2.0 was noted for
S. rimosus, which displayed poor growth with only several
irregular and barely visible biomass fragments found within
the ask volume (Fig. 1b). These structures could have been
developed due to slow biomass build-up on hyphal aggre-
gates transferred from the agar slants during inoculation. In
A. terreus vs. S. rimosus cocultures (Fig. 1c) the observed
structures resembled the typical agglomerates of A. terreus,
with dense and compact cores. Although not ideally spheri-
cal they were classied as pellets, as opposed to the dense
clumps recorded in the A. terreus axenic cultures at pH
2.0. Arguably, the presence of S. rimosus spores promoted
the formation of more typical A. terreus-like pellets in the
coculture despite the low pH level. Hardly any traces of S.
rimosus laments were observed in the coculture.
At the initial pH 3.0, the formation of pellets containing
a clearly visible core was recorded for A. terreus (Fig. 1a).
Therefore, relative to the A. terreus culture initiated at pH
2.0, the morphological transition from clumps to pellets
took place in the axenic A. terreus variant in response to
the initial pH of 3.0. A drastic change with respect to pH
2.0 was also noted for S. rimosus axenic cultures, which
at pH 3.0 displayed the growth in the form of macromor-
phological structures, i.e. large (bigger than 1 cm), smooth,
olive-shaped biomass agglomerates (Fig. 2b). Only 2 or 3
structures of this type were formed within a single ask
(210 mL), depending on the experimental replicate. As far
as the coculture at pH 3.0 was concerned, the observed mor-
phology closely resembled the one recorded in the axenic
fungal counterpart, with the visible formation of A. terreus-
like pellets (Fig. 1c).
Since the transition from clumped to pelleted growth
took place in the pH 3.0 variant of A. terreus axenic culture,
the formation of fungal pellets was expected to occur within
the pH interval between 4.0 and 5.9. Surprisingly, at pH 4.0
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Archives of Microbiology (2024) 206:452
Fig. 1 The microscopic images of morphological forms in A. terreus
axenic culture (a), S. rimosus axenic culture (b), and A. terreus vs.
S. rimosus coculture (c). The samples were drawn after 168 h of cul-
tivation. The cultivations were performed in the medium containing
glucose, lactose, yeast extract, KH2PO4, MgSO4 ∙ 7 H2O, ZnSO4 ∙ 7
H2O, NaCl, Fe(NO)3 ∙ 9 H2O, biotin, and trace element solution. The
microorganisms were propagated in 500 mL at-bottom glass asks.
The cultivation was carried out in the laboratory shaker at 28 °C and
120 min− 1. The macromorphological forms, too large to be observed
by using the microscope, are not depicted here. Their photographic
images are presented in Fig. 2
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Archives of Microbiology (2024) 206:452
and 5.9. Another fungal SM identied in the broth, namely
butyrolactone I, was found exclusively in the axenic culture
at pH 5.9 (Fig. 4d). All the identied SMs of A. terreus were
previously reported to be produced in A. terreus vs. S. rimo-
sus cocultures (Boruta et al. 2021, 2024).
Final pH
Immediately after the completion of cultivation, the pH val-
ues were determined for the investigated cultures (Fig. 5).
The statistically signicant dierences between the cocul-
tures and the corresponding axenic cultures (P = 0.046 and
0.017 calculated with respect to A. terreus and S. rimosus
axenic variants, respectively) were recorded only in the case
of pH 5.0, with the mean nal pH values equal to 5.11, 5.26
and 4.18 for the axenic culture of A. terreus, axenic culture
of S. rimosus and the coculture, respectively. So, regarding
the process which was started at pH 5.0, the non-negligible
decrease in pH due to microbial cocultivation was observed
compared to the axenic variants.
Discussion
The inuence of pH on the production of microbial SMs
is associated with the pH preference of a given species in
terms of biomass growth, and the biosynthetic pathway-spe-
cic eect of pH on the biosynthesis of a given SM (Bra-
khage 2013; Keller 2019). In cocultures, there are additional
factors that contribute to the bioprocess outcome, e.g., the
interspecies interactions and the dierences among the
cocultivated species with regard to their pH preferences. If
the pH of the medium lies within the growth-promoting pH
range of a given microorganism, the chances of survival of
this microbe are higher than at less favorable pH levels. Of
course, the pH is merely one of the factors that shape the
outcome of microbial cocultivation, such as medium com-
position, inoculation time, inoculation ratio, and bioprocess
conditions (Kapoore et al. 2022). Here, at pH levels 2.0,
3.0 and 4.0 the growth of S. rimosus biomass in the cocul-
tures was not detectable, while the pellets closely resem-
bling the morphological forms of A. terreus were developed
(Fig. 1c). The opposite scenario was noted for the pH 5.9
variant, which resulted in the formation of S. rimosus-like
pellets and no traces of A. terreus structures. Apparently, the
pH 5.0 coculture was characterized by the parallel develop-
ment of both species, yet the sizes of the A. terreus-like and
S. rimosus-like morphological forms were smaller than in
the axenic counterparts (Fig. 1a, b). Clearly, the lower pH
values (i.e., 2.0, 3.0, and 4.0) favored the growth of fungal
biomass at the cost of S. rimosus, while at pH 5.9 the acti-
nomycete exerted its dominance and practically eliminated
the analytical standard (Fig. 3a), and the closely related
metabolite 2-acetyl-2-decarboxamido-oxytetracycline,
abbreviated as ADOTC (Fig. 3b). In addition, 4 molecules
putatively assigned to the rimocidin biosynthetic family
(Boruta et al. 2021) were found in the coculture, namely
rimocidin (Fig. 3c), CE-108 (Fig. 3d), rimocidin (27-ethyl)
(Fig. 3e), and oxidized rimocidin, i.e., the metabolite result-
ing from the elimination of “CH2O2” moiety from rimocidin
(Fig. 3f). Among the identied S. rimosus products, 5 mol-
ecules were found both in the cocultures and the axenic cul-
tures (Fig. 3a-e), whereas one of the rimocidin derivatives,
namely oxidized rimocidin, was detected exclusively under
coculture conditions. Importantly, the production of all S.
rimosus metabolites occurred only in the variants initiated
at pH equal to 5.0 and 5.9 (Fig. 3). In other words, the SM
biosynthetic activity was blocked at the pH values of 2.0,
3.0, and 4.0 regardless of the product or biosynthetic family.
Considering the levels of SMs, no statistically signicant
dierences between the cocultures and axenic cultures were
observed for ADOTC (Fig. 3b), while for oxytetracycline
(Fig. 3a) and the rimocidins (Fig. 3c, d, e) the outcome
was clearly dependent on the initial pH level. With regard
to the rimocidin family of molecules (Fig. 3c, d, e), their
levels in axenic cultures at pH 5.0 were far higher than in
the cocultures. By contrast, at pH 5.9 the production levels
in cocultures and axenic variants did not exhibit signicant
dierences (Fig. 3c, d, e). In the case of oxytetracycline,
the modest production improvement (P = 0.048) was noted
in the coculture relative to the axenic culture, but solely at
pH 5.9 (Fig. 3a). At pH 5.0, the levels of oxytetracycline
reached in coculture did not show signicant dierences
compared with the axenic culture.
Regarding the SMs produced by A. terreus, their presence
was noted solely in the axenic variants (Fig. 4). This group
included mevinolinic acid (i.e., lovastatin β-hydroxy acid),
which was quantitatively assayed by using a standard solu-
tion (Fig. 4a), as well as the closely related and putatively
identied molecules 3α-hydroxy-3,5-dihydromonacolin L
(Fig. 4b) and 4a,5-dihydromevinolinic acid (Fig. 4c). These
3 metabolites, representing the statin biosynthetic pathway
in A. terreus, were detected in the axenic cultures at pH 5.0
Fig. 2 The photographic images of morphological forms in A. terreus
axenic culture at the initial pH 4.0 (a), S. rimosus axenic culture at the
initial pH 3.0 (b), and S. rimosus axenic culture at the initial pH 4.0
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Archives of Microbiology (2024) 206:452
Fig. 3 The levels of secondary metabolites produced in axenic cultures
of S. rimosus and the cocultures of A. terreus and S. rimosus at dier-
ent initial pH values, namely oxytetracycline (a), putative 2-acetyl-
2-decarboxamido-oxytetracycline (ADOTC) (b), putative rimocidin
(c), putative CE-108 (d), putative rimocidin (27-ethyl) (e) and puta-
tive oxidized rimocidin (f). The levels of secondary metabolites were
determined after 168 h of cultivation. The secondary metabolites in A.
terreus vs. S. rimosus cocultures were identied previously (Boruta
et al. 2021, 2024). The results are given as the mean ± SD (n = 3). *,
P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not signicant
1 3
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Archives of Microbiology (2024) 206:452
this eect was recorded solely at pH 5.9. By contrast, for the
coculture initiated at pH 5.0 the results were comparable
with the levels noted for the axenic counterpart. In the case
of rimocidin (Fig. 3c) and CE-108 (Fig. 3d), one could see
their production being signicantly inhibited due to coculti-
vation but only at pH 5.0. Based on these results, it is clear
that the investigation of cocultivation-related eect on SM
production need to be performed at various pH values to
get a comprehensive view on this matter. In addition, one
must be aware that the examination of pH eect on the SM
production in axenic cultures does not necessarily reect the
correlations observed for the coculture counterparts.
To the best of our knowledge, this is the rst report on the
inuence of pH on the morphological forms of A. terreus
(Figs. 1a and 2a) and S. rimosus (Figs. 1b, 2b and c). The
axenic growth of S. rimosus was barely visible at pH 2.0,
the fungus from the coculture. At pH 5.0, there was no clear
dominance of any of the species, as they both coexisted in
the coculture. Yet, the fungal SMs were not produced at pH
5.0 (Fig. 4) despite the presence of fungal-like morphologi-
cal forms. Since the axenic variants of A. terreus at pH 5.0
did exhibit the production of SMs, the lack of fungal SMs in
the coculture was evidently due to the presence of S. rimo-
sus. As the growth of A. terreus was indeed observed in the
coculture initiated at pH 5.0, the inhibition seemed to occur
at the level of fungal secondary metabolic pathways rather
than the growth-related primary pathways.
Most importantly, the eect of cocultivation of the pro-
duction of a given SM (relative to the axenic culture) was
shown to be dependent on the initial pH level. For example,
the biosynthesis of oxytetracycline (Fig. 3a) was slightly
enhanced in coculture relative to the axenic culture, however,
Fig. 4 The levels of A. terreus secondary metabolites produced at
dierent initial pH values, namely mevinolinic acid (a), putative
3α-hydroxy-3,5-dihydromonacolin L (b), putative 4a,5-dihydrome-
vinolinic acid (c), and butyrolactone I (d). The levels of secondary
metabolites were determined after 168 h of cultivation. The second-
ary metabolites in A. terreus vs. S. rimosus cocultures were identi-
ed previously (Boruta et al. 2021; 2024). The results are given as the
mean ± SD (n = 3). *, P < 0.05; ns, not signicant
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Archives of Microbiology (2024) 206:452
morphological characteristics. While the axenic cultures
yielded large, macromorphological biomass agglomerates
of various shapes and sizes, the growth in the coculture
took the pelleted, A. terreus-like form. It is known that the
agglomeration of Aspergillus spores strongly depends on
culture pH, which in turn aects the spore surface charges
(Grimm et al. 2005). Possibly, the formation of fungal-like
pellets in the coculture may have taken place due to the
charge shielding eect exhibited by S. rimosus spores on the
conidia of A. terreus, what led to the agglomeration eect
being distinct from the one observed in the fungal axenic
culture. Presumably, the pH level of 4.0 favored the growth
of A. terreus and its dominance over the actinomycete, what
ultimately led to the development of pellets that resembled
A. terreus rather than S. rimosus growth (Fig. 1c).
The coculture initiated at pH 5.0 was exceptional
among the tested variants. Firstly, it was the only cocul-
ture in which both the A. terreus-like and S. rimosus-like
morphological forms were recorded in the broth (Fig. 1c).
Secondly, the nal pH of this coculture was visibly lower
than in the corresponding axenic cultures (Fig. 5). Thirdly,
despite the presence of A. terreus-like pellets the coculture
occurred in the form of large irregular biomass agglomer-
ates at pH 3.0 and 4.0, and, nally, manifested itself through
the formation of pellets at pH 5.0 and 5.9. In the case of
A. terreus axenic culture, there was a clumped and pel-
leted growth at pH 2.0 and 3.0, respectively. At pH 4.0, the
micromorphological forms were replaced by macromorpho-
logical ones, i.e. large irregular biomass agglomerates. At
higher pH values, i.e., 5.0 and 5.9, the pelleted growth was
again observed. The morphological similarities and dier-
ences between S. rimosus and A. terreus were evident. Both
species displayed the pelleted growth at pH 5.0 and 5.9 and
the development of macromorphological biomass agglom-
erates at pH 4.0. The dierences were, however, recorded
at lower pH values. At pH 2.0, the growth of the actinomy-
cete was poor (only the traces of biomass were visible in the
ask), unlike the growth of A. terreus, which propagated
in the form of clumps. At pH 3.0, S. rimosus formed mac-
romorphological spherical biomass agglomerates (with the
diameters exceeding 1 cm), whereas the fungus proliferated
as pellets characterized by well-dened, distinct cores.
At pH 4.0, the coculture (Fig. 1c) diered markedly from
its axenic counterparts (Fig. 2a, c) in terms of the displayed
Fig. 5 The nal pH values (measured after 168 h of cultivation) in the A. terreus and S. rimosus axenic cultures and cocultures initiated at dierent
pH values. The results are given as the mean ± SD (n = 3). *, P < 0.05; ns, not signicant
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Archives of Microbiology (2024) 206:452
the fungus still managed to exert the eect on the actino-
mycete from within the pellets. The origin of the rimocidin
derivative could be associated with the biotransformation
activity of the fungus, as previously suggested (Boruta et al.
2021), but its de novo biosynthesis by S. rimosus in response
to the stimulus provided by A. terreus cannot be excluded.
In a previous study, Wargenau et al. (2011) investigated
the pH-dependent electrostatic surface potential of Asper-
gillus niger conidia under acidic conditions. It was pro-
posed that the surface potential depends on the release of
melanin and the addition of negative charges to the outer
layer of the spores. Importantly, the results indicated that
the melanin release occurring under acidic pH is associ-
ated with the deprotonation of carboxyl groups bound to
the pigment layer of the spores. The authors concluded that
the pH-dependent repulsion between fungal spores depends
on a number of factors, including the ionic strength of the
medium, the thickness of surface coating, and the properties
of individual ions present in the system. Since the events of
spore agglomeration result from the net eect of repulsion
and attraction between the spores, the tendency of a given
species to form pellets cannot be attributed solely to the pH
level. In the present study, the co-inoculation of A. terreus
and S. rimosus spores at pH 4.0 resulted in the morphologi-
cal outcome (i.e., the fungal-like pellet formation) that did
not resemble the macromorphological structures recorded
in the corresponding axenic variants. So, the presence of S.
rimosus spores at pH 4.0 altered the agglomeration of A. ter-
reus conidia, leading to the formation of pellets which were
not seen in the corresponding axenic cultures. As already
mentioned in the discussion, the relatively low pH appar-
ently favored the growth of A. terreus at the cost of S. rimo-
sus, so the observed macromorphological outcomes in the
coculture initiated at pH 4.0 was the formation of A. terreus-
like (not S. rimosus-like) pellets.
In the previous study, Bizukojć et al. (2012) demon-
strated that relatively small amounts of mevinolinic acid
can be found in the A. terreus axenic cultures initiated at
pH values even as low as 3.5 and 4.5. In the present work,
there was no evidence of mevinolinic acid presence in the
variants initiated at pH less than 5.0. The discrepancy can
be attributed to a large number of bioprocess-related dier-
ences between the two studies. Here, the composition of the
growth medium included glucose and lactose as the sources
of carbon, while glucose was absent from the medium used
by Bizukojć et al. (2012). The previous study was based on
the use of 24-h precultures, while the variants investigated
in the present work were inoculated by applying the spore
suspensions. In addition, the speed of the laboratory shaker,
the cultivation temperature, the duration of the bioprocess,
and the culture working volume used here diered from the
ones reported by Bizukojć et al. (2012). All these factors
yielded no traces of A. terreus products (Fig. 4), what indi-
cated the inhibition of fungal biosynthetic machinery due to
S. rimosus dominance. Finally, it led to a marked inhibition
in terms of the production of rimocidin and its derivatives
compared with the S. rimosus axenic culture (Fig. 3c, d, e).
At the same time, the coculture-related inhibition of oxytet-
racycline (Fig. 3a) and ADOTC (Fig. 3b) biosynthesis at pH
5.0 was less evident, with no statistically signicant dier-
ence observed between the coculture and the axenic culture.
In other words, at pH 5.0 the inuence of cocultivation on
the production of rimocidins was not comparable with the
eect exerted on oxytetracycline and ADOTC formation,
i.e., the response of SM biosynthetic route to cocultivation
at a given pH varied among the pathways.
The study demonstrated that biomass growth alone is
insucient to predict SM production, regardless if the bio-
process is carried out under the axenic or coculture con-
ditions. The biosynthesis of SMs in axenic cultures was
recorded solely at pH 5.0 and 5.9, while the axenic growth
of A. terreus and S. rimosus took place across the investi-
gated pH range, with the only visible growth inhibition seen
for S. rimosus at pH 2.0. In the cocultures, the presence of A.
terreus-like morphological structures did not lead to fungal
SM production, even if the A. terreus-like and S. rimosus-
like forms coexisted (i.e., at pH 5.0). By contrast, the SMs
of S. rimosus were detected in the broth whenever the S.
rimosus-like pellets were formed in the coculture, i.e., at pH
5.0 and 5.9. Apparently, the eect of fungal growth at pH
5.0 was strong enough to decrease (but not eliminate) the
rimocidin production by S. rimosus in the coculture, but it
failed to block the actinomycete biomass development and
oxytetracycline biosynthesis. At the same time, the growth
of S. rimosus at pH 5.0 did not eliminate A. terreus biomass
from the coculture, but it did block the production of detect-
able levels of all fungal SMs. So, the actinomycete seemed
to exert a stronger SM production-blocking eect on the
fungus than vice versa. As already mentioned, achieving the
coexistence of two species in the coculture is clearly insuf-
cient to ensure the SM production by any of them.
The lack of visible A. terreus-like pellets in the coculture
initiated at pH 5.9 did not mean that the fungus had no inu-
ence on the coculture outcome, as indicated by the presence
of the oxidized rimocidin, which was recorded exclusively
under cocultivation conditions. It should be mentioned that
this product was also seen in the coculture initiated at pH
5.0, a variant in which the fungal-like pellets were clearly
observable. It means that the “A. terreus vs. S. rimosus”
cocultures which were started at pH 5.0 or 5.9 yielded the
rimocidin derivative regardless if the A. terreus-like pellets
were formed (as in the “pH 5.0” coculture variant) or not
(as in the pH “5.9” variant). Perhaps the spores of A. terreus
were engulfed within the pellets of S. rimosus at pH 5.9, and
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Archives of Microbiology (2024) 206:452
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Author contributions TB: Conceptualization, Methodology, Valida-
tion, Formal analysis, Investigation, Visualization, Supervision, Writ-
ing - Original Draft, Writing - review & editing. MF: Formal analy-
sis, Investigation, Visualization. WP: Formal analysis, Investigation,
Visualization. GE: Formal analysis, Investigation, Visualization. MB:
Methodology, Investigation, Writing - review & editing.
Funding The project was funded by the National Science Centre (Re-
public of Poland) (grant number 2017/27/B/NZ9/00534).
Data availability No datasets were generated or analysed during the
current study.
Declarations
Competing interests The authors declare no competing interests.
Open Access 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) and the
source, provide a link to the Creative Commons licence, and indicate
if changes were made. The images or other third party material in this
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