Journal of Applied Microbiology ISSN 1364-5072

REVIEW ARTICLE

Microbial metabolomics: essential definitions and the

importance of cultivation conditions for utilizing Bacillus
species as bionematicides
I. Horak1, G. Engelbrecht1, P.J. Jansen van Rensburg2 and S. Claassens1
1 Unit for Environmental Sciences and Management, North-West University, Potchefstroom, South Africa
2 Focus Area: Human Metabolomics, North-West University, Potchefstroom, South Africa

Keywords
Bacillus, bionematicide, enzymes, metabolite,
metabolomics, root-knot nematode.
Correspondence
Sarina Claassens, Unit for Environmental
Sciences and Management, North-West
University, Potchefstroom, South Africa.
E-mail: sarina.claassens@nwu.ac.za
2018/2339: received 13 December 2018,
revised 4 February 2019 and accepted 4 Feb-
ruary 2019
doi:10.1111/jam.14218
Abstract
Root-knot nematodes are destructive phytopathogens that damage agricultural
crops globally, and there is growing interest in the use of biocontrol based on
rhizobacteria such as Bacillus to combat Meloidogyne species. It is hypothesized
that nematicidal activity of Bacillus can be attributed to the production of
secondary metabolites and hydrolytic enzymes. Yet, few studies have
characterized these metabolites and their identities remain unknown. Others are
speculative or fail to elaborate on how secondary metabolites were detected or
distinguished from primary metabolites. Metabolites can be classified based on
their origin as either intracellular or extracellular and based on their function, as
either primary or secondary. Although this classification is in general use, the
boundaries are not always well defined. An understanding of the secondary
metabolite and hydrolytic enzyme classification of Bacillus species will facilitate
investigations aimed at bionematicide development. This review summarizes the
significance of Bacillus hydrolytic enzymes and secondary metabolites in
bionematicide research and provides an overview of known classifications. The
importance of appropriate cultivation conditions for optimum metabolite and
enzyme production is also discussed. Finally, the use of metabolomics for the
detection and identification of nematicidal compounds is considered.

According to Arthurs and Dara (2018), a total of 356

Introduction
Biocontrol agents are considered an environmentally
friendly alternative to the use of chemical pesticides
against phytopathogens such as the root-knot nematode
(RKN) genus Meloidogyne. Biological control agents or
biopesticides refer to natural products derived from ani-
mals, plants or micro-organisms (Arthurs and Dara 2018)
that are applied as natural antagonist species to combat
plant pathogens (P
erez-Garc
ıa et al. 2011). When com-
pared to chemical pesticides, the majority of biopesticides
have a reduced risk (Arthurs and Dara 2018) due to their
specific antagonism towards target pests, biodegradability
in the environment (Berini et al. 2018), and high effi-
ciency (Ben Abdallah et al. 2018). These qualities are very
advantageous, and their environmental safety enables
application close to harvesting time (Mnif and Ghribi
2015).
active biopesticide ingredients for use against mites,
insects and nematodes are registered in the United States
of America. Of these 356 ingredients, 57 are of microbial
origin. This highlights the significance of micro-organ-
isms in biological control, which can be achieved by
directly adding the living antagonistic strain as an inocu-
lum to the soil. Alternatively, a combination of microbial
enzymes and metabolites from biocontrol strains can be
formulated, produced and optimized. This approach will
overcome certain limitations in the use, maintenance and
storage of living organisms (Berini et al. 2018).
Although many classes of bacterial metabolites and
enzymes are known, not all of the compounds present
within these classes have been studied to determine their
nematicidal potential. To determine the identity of the
metabolites and/or enzymes responsible for the nematici-
dal activity demonstrated by bacterial species, researchers

326 Journal of Applied Microbiology 127, 326--343 © 2019 The Society for Applied Microbiology
I. Horak et al.
must first investigate previously classified compounds
before attempting to identify novel nematicidal com-
pounds. One such class of metabolites of which the
nematicidal activity is not clearly known are secondary
metabolites. It is important to know with absolute cer-
tainty whether the reported nematicidal compounds are
in fact secondary metabolites or primary metabolites or
even other compounds. The reason for this is to ensure
that the bacterial cultures of interest are incubated and
sampled according to the growth phase that needs to be
reached for production of the different classes of com-
pounds. In other words, for the detection of secondary
metabolites the bacterial cultures should be sampled dur-
ing the stationary phase of growth during which sec-
ondary metabolites are produced. If the bacterial cultures
are sampled too early or too late, secondary metabolites
of importance might not have been produced yet or
might have already been degraded, re-utilized or con-
verted into other compounds. Scientists must build on
existing knowledge to further advance bionematicide
research. Therefore, the purpose of this review is to: (i)
establish a clear definition that will differentiate between
primary and secondary metabolites in the context of
metabolomics studies; (ii) give an overview of known
Bacillus secondary metabolites and enzymes with nemati-
cidal potential; (iii) highlight the importance of cultiva-
tion conditions for optimum metabolite and enzyme
production; and (iv) discuss the use of metabolomics for
the detection of possible nematicidal compounds. For the
purpose of this review, the authors have focused on the
biocontrol of the Meloidogyne genus (RKNs), due to their
worldwide distribution (Caboni et al. 2013), wide host
range and predominance as nematode pests in South
African cereal (maize, grain sorghum, millet, barley, rye
and oat; McDonald et al. 2017) and vegetable (soybean,
groundnut, potatoes, tomatoes, onions and sugarcane)
crops (Berry et al. 2017; Fourie et al. 2017; Jones et al.
2017).
Biological control based on Bacillus species
The rhizosphere is considered a rich source of micro-
organisms that produce a multitude of bioactive mole-
cules, and it is estimated that the concentration of rhizo-
sphere bacteria is 10-1000 times higher than bacterial
populations found in soil (Gouda et al. 2018). Many
nematophagous rhizobacteria, including Pasteuria spp.,
Pseudomonas spp. and Bacillus spp., have been used for
the biological control of phytopathogens (Berini et al.
2018). Since Bacillus spp. are directly associated with
plants and the soil environment, the inhibition of phy-
topathogens is a fundamental function of the bioactive
molecules they produce (Mongkolthanaruk 2012). These
Journal of Applied Microbiology 127, 326--343 © 2019 The Society for Applied Microbiology
Bionematicides from Bacillus
soil-dwelling bacteria are highly suitable for use as bione-
maticides since they also show rapid growth and produc-
tivity during cultivation (Niu et al. 2006).
There is particular interest in Bacillus spp. especially in
agriculture applications as some demonstrate the ability
to control plant-parasitic nematodes (PPNs; P
erez-Garc
ıa
et al. 2011), and more specifically the Meloidogyne genus
(Abbasi et al. 2014; Zhao et al. 2018). The majority of
products commercially available for the promotion of
plant growth and biological control consist of Bacillus
subtilis and Bacillus amyloliquefaciens (Aleti et al. 2015).
Bacillus spp. are classified as extracellular plant growth-
promoting rhizobacteria inhabiting the rhizosphere or the
spaces between the root cortex cells (Gouda et al. 2018)
and the majority are regarded as safe micro-organisms
(Frikha-Gargouri et al. 2017). This is demonstrated by
the United States Food and Drug Administration award-
ing B. subtilis the status of ‘generally regarded as safe’
(Cawoy et al. 2011). This designation implies that B. sub-
tilis cannot cause harm, disease or death to another
organism (nonpathogenic) and is therefore safe for both
humans and the environment. Bacillus spp. have great
potential as biopesticides (Ramezani Moghaddam et al.
2014; Gao et al. 2016) due to the combination of benefi-
cial traits they exhibit. They are ubiquitous within agri-
cultural soil (Gardener 2004), have the ability to thrive
under various environmental conditions and can survive
in different habitats due to their ability to produce an
overabundance of antimicrobial compounds (Abriouel
et al. 2011). Additionally, they produce spores which are
remarkably resistant (Ongena and Jacques 2008), and
they readily colonize plant roots (Jamal et al. 2017) and
are known to promote plant growth (Abbasi et al. 2014;
Lee and Kim 2016). This combination of traits is ideal as
micro-organisms exhibiting a variety of beneficial attri-
butes can be utilized in the development of biological
pesticides (Ben Ayed et al. 2014).
Bacillus also display taxonomic and metabolic diversity
(Hamdache et al. 2011), and one agriculturally important
attribute is their ability to produce nematicidal com-
pounds which can include secondary metabolites such as
antibiotics, cyclic lipopeptides, polyketides (PKs) and
bacteriocins (Fig. 1). A classification of reported Bacillus
secondary metabolites based on their mode of synthesis
(ribosomally or nonribosomally) is shown in Fig. 1. An
estimated 795 secondary metabolite antibiotics are pro-
duced by Bacillus species (Awad et al. 2012). Antibiotic
peptides are the most thoroughly studied and have
important applications in medicine. A variety of other
secondary metabolites are also produced by this genus
with B. subtilis and B. amyloliquefaciens contributing a
considerable part of the observed Bacillus lipopeptide and
polyketide diversity (Aleti et al. 2015). This is evidenced
327
Bionematicides from Bacillus
Secondary metabolites
Ribosomally synthesized
Other Bacteriocins Peptide
antibiotics
Lantibiotics
• Lichenin
• Megacin
• Cerein
• Thuricin • Subtilin
• Subtilosin A
• Lichenicidin
• Amylolysis
• Ericin
• Mersacidin
• Entianin
• Subtilomycin
• Formicin
• Cerecidin
• Sublancin
• Mejucin
Figure 1 Classification of reported Bacillus secondary metabolites.
by B. subtilis which devotes 4% of its genome to the syn-
thesis of PKs, nonribosomal peptides, bacteriocins and
various antibiotics (Fickers 2012). According to Aleti
et al. (2015), the low level or complete absence of polyke-
tide and lipopeptide production in genera inhabiting
other environments (e.g. water) is indicative of the
important role that these secondary metabolites play in
plant-associated habitats.
Although the majority of Bacillus species produce sec-
ondary metabolites, their production is not always con-
served among Bacillus species or even strains. Palazzini
et al. (2016) constructed a phylogenomic neighbour-join-
ing tree from the core genomes of Bacillus velezensis, B.
amyloliquefaciens and B. siamensis strains. All of the
strains possessed gene clusters for the production of sur-
factin, bacillibactin, bacillaene, amylocyclicin and an
iturin-like substance (iturin or bacillomycin), while all
the strains of B. valezensis contained bacilysin, fengycin,
macrolactin and difficidin gene clusters (Palazzini et al.
2016). The genomes of B. subtilis and B. amyloliquefaciens
also contain genes for the production of surfactin, iturin,
fengycin and kurstakin (Aleti et al. 2015). Genome-
mining was used with the aim of extending the character-
ization of B. velezensis RC 218, and results indicated that
nine secondary metabolites were conserved in all strains
328 Journal of Applied Microbiology 127, 326--343 © 2019 The Society for Applied Microbiology
I. Horak et al.
Nonribosomally synthesized
Polyketides Peptide Cyclic Lipopeptides
antibiotics
Surfactin
• Bacillaene
• Difficidin
• Macrolactin • Surfactin A
• Aurantinins • Surfactin B
• Basiliskamide • Surfactin C
Iturin
• Iturin A • Bacillomycin LC
• Iturin C • Bacillomycin DC
• Bacillomycin D • Mycosubtilin
• Bacillomycin F
Fengycin
Kurstakin
and one unique secondary metabolite, ericin, was
detected. This was the first study to confirm the produc-
tion of ericin by this strain (Palazzini et al. 2016).
Many studies have demonstrated the different biologi-
cal activities exhibited by Bacillus metabolites or enzymes,
including antifungal (Arroyave-Toro et al. 2017; Gu et al.
2017; Salazar et al. 2017), antibacterial (Fan et al. 2017)
and nematicidal (Oliveira et al. 2007; Mendoza et al.
2008; Borah et al. 2018). Antifungal activity has been
investigated most frequently, and since various Bacillus
metabolites are known to exhibit antifungal or fungicidal
activity, this implies the possibility of nematicidal activity
from the same metabolites. Metabolites with antifungal
activity could also hold potential as bionematicides as
these biological activities are not always restricted to a
single domain of life (bacteria, archaea and eukaryotes).
This was demonstrated by Ramyabharathi et al. (2018)
who investigated the biocontrol potential of B. subtilis
Bbv 57 (KF718836) against a wilt-RKN disease complex
consisting of Fusarium oxysporum f. sp. gerberae
(KM523669) and Meloidogyne incognita. Culture filtrates
of the bacterium resulted in both the inhibition of myce-
lial growth of F. oxysporum and lethality towards M.
incognita eggs and juveniles. The presence of the lipopep-
tides, surfactin and iturin was confirmed (Ramyabharathi
I. Horak et al.
et al. 2018). Likewise, Adam et al. (2014) investigated the
use of B. subtilis as possible ‘multi-purpose’ bacteria for
the control of both fungal pathogens and RKNs. It is
known that B. subtilis is a fungal antagonist, but was able
to induce systemic resistance in tomato plants for the
control of M. incognita. For the purpose of this review,
the authors have focused on the nematicidal potential of
Bacillus secondary metabolites and hydrolytic enzymes.
Mendoza et al. (2008) evaluated the nematicidal activ-
ity of Bacillus firmus cell-free culture filtrates against three
agriculturally important types of nematodes (burrowing,
root-knot and stem nematode). These pure culture fil-
trates resulted in significant nematode paralysis, mortality
and reduced egg hatching (Mendoza et al. 2008). Second-
ary metabolites produced by B. firmus during fermenta-
tion were assumed to be responsible for the reduction in
hatching of M. incognita eggs. Similar results were
reported by Xiong et al. (2015). Metabolites produced by
Bacillus cereus and B. subtilis exhibiting nematicidal activ-
ity against the plant-parasitic nematode, Meloidogyne
exigua, were identified as uracil, 9H-purine and dihy-
drouracil (Oliveira et al. 2014). Results demonstrated that
dihydrouracil could possibly control RKNs more effi-
ciently than the chemical nematicide carbofuran, since it
had a lower LC50 value than carbofuran (Oliveira et al.
2014). A study by Yanfei et al. (2011) aimed to evaluate
the efficacy of B. subtilis as a bionematicide against four
nematode species, including Meloidogyne javanica. It was
established that the purL gene, encoding a 50 -phosphori-
bosyl-N-formyl-glycinamidine (FGAM) synthase II, was
associated with the nematicidal activity of B. subtilis.
Microbial metabolites
Metabolites are chemical compounds of a low molecular
weight (<1000 Da) that play a critical role in microbial
metabolism (chemical conversion; Pinu et al. 2017). Col-
lectively, all the metabolites of a specific micro-organism
are referred to as its metabolome (Aldridge and Rhee
2014). The word ‘metabolome’ was first proposed in
1997 (Beale et al. 2016), and it can be divided into three
distinct matrices: within the cell, in the extracellular med-
ium, and in the culture headspace. The metabolome is
indicative of an organism’s biological function as it is
most closely related to the phenotype (Macintyre et al.
2014). Metabolites are characterized by infinite half-lives
and are thus taken up, produced, degraded or excreted
(Villas-B^oas et al. 2007). However during metabolic
activities, metabolites are not only formed but also con-
verted into different compounds. The difference between
the rate of metabolite formation and conversion is
responsible for the amount of each metabolite present
within a microbial cell (Pinu et al. 2017).
Journal of Applied Microbiology 127, 326--343 © 2019 The Society for Applied Microbiology
Bionematicides from Bacillus
Generally, there are three types of metabolites present
in biological samples which can be found both intracellu-
larly and extracellularly: (i) water soluble (polar), (ii)
water insoluble (nonpolar) and (iii) volatiles (Villas-B^oas
et al. 2007). Although metabolites are highly diverse, the
diversity is at the level of atoms and not molecules
(Aldridge and Rhee 2014), and metabolites can be classi-
fied based on their origin as either endogenous or exoge-
nous and on their function as either primary or
secondary. Endogenous metabolites originate from within
a cell, while exogenous metabolites arise from outside the
cell (Vaidyanathan 2005). Depending on which growth
phase the microbial cells are in, either primary or sec-
ondary metabolites will be produced. The metabolic path-
ways of primary metabolites are associated with the
production (anabolic activity) and breakdown (catabolic
activity) of metabolites, while the metabolic pathways of
secondary metabolites are associated with low growth rate
and a response to stress (Pinu et al. 2017). Although the
abovementioned classifications of metabolites are used
throughout the literature, the boundaries between them
are not well defined and are thus open to interpretation
(Vaidyanathan 2005). For the purpose of this review, the
authors have distinguished between primary and sec-
ondary metabolites based on function and between intra-
cellular and extracellular metabolites based on origin.
During bacterial growth, primary metabolites are syn-
thesized continuously. They are essential for survival and
are required for important cellular processes such as
growth, development and reproduction. When micro-
organisms cannot produce these vital metabolites, aux-
otrophy will result (Pande and Kost 2017) and the organ-
ism will die unless the metabolite is exogenously
supplied. Primary metabolites are conserved across phyla
and kingdoms (Karlovsky 2010), and as a result, primary
metabolites produced by the majority of microbial species
are similar (Vaidyanathan 2005). They are usually found
in low quantities within the cell due to their fast turnover
rates. As a result, the levels of primary metabolites within
the cell are generally lower than the levels of secondary
metabolites within the cell; however, this is not always
the case as intermediate metabolites from the primary
metabolism can be produced in abundance by micro-
organisms and might then be excreted into the extracellu-
lar medium (Pinu et al. 2017).
As opposed to primary metabolites, secondary metabo-
lites are not essential for the survival of the producing
organisms and are not required for normal growth and
development. Rather, they provide the producing organ-
isms with a survival advantage such as improving the
nutrients available for uptake or protection against envi-
ronmental stressors (Breitling et al. 2013) and are thus
considered dispensable (Sansinenea and Ortiz 2011).
329
Bionematicides from Bacillus I. Horak et al.

Nutrient deprivation usually induces secondary metabo-
lism (Ruiz et al. 2010), and secondary metabolites are
thus produced during the stationary phase of bacterial
growth when nutrients become depleted and waste accu-
mulates. Consequently, it is hypothesized that metabolites
produced during this growth phase will exhibit higher
biological activity. This was evidenced in a study by Ayed
et al. (2015) who demonstrated that the antimicrobial
activity of bacteriocins produced by B. amyloliquefaciens
An6 reached a maximum early in the stationary phase.
Secondary metabolites are either synthesized ribosomally
(such as bacteriocins) or nonribosomally (such as PKs),
and the genes that encode the enzymes for their synthesis
are usually chromosomal (Ruiz et al. 2010). Moreover,
they exhibit higher accumulation levels than primary
metabolites (Covington et al. 2017) and either accumu-
late within microbial cells or are released into the extra-
cellular medium since they are substrates of only a few
metabolic reactions (Pinu et al. 2017). Although distinctly
different in the roles they fulfil, primary metabolism and
secondary metabolism are closely related as primary
metabolism provides the energy and precursor molecules
required for secondary metabolism (Villas-B^ oas et al.
2007).
Microbial secondary metabolism is a rich source of
bioactive molecules (Barkal et al. 2016), and the meta-
bolic pathways responsible for the production of aromatic
compounds, isoprenes, peptides and PKs are some of the
main biochemical pathways of secondary metabolism
(Ruiz et al. 2010). Tyc et al. (2017) recently reviewed the
two main secondary metabolite classes of soil bacteria,
namely volatile organic compounds (VOCs) and soluble
metabolites. This classification is based on polarity and
the evaporation and diffusion abilities of the metabolites.
VOCs can evaporate and diffuse through pores filled
either with water or air, whereas soluble metabolites are
water soluble due to their high polarity and exhibit more
distinctive bioactivities. Bacteriocins, lipopeptides and
PKs are soluble secondary metabolites, while pyrazines,
indole and certain sulphur-containing compounds repre-
sent the VOCs. A bacterial volatile of particular interest is
dimethyl disulphide. It has been approved by the United
States Environmental Protection Agency and is used as a
fumigant against nematodes and soil-borne pathogens
under the commercial appellation of Palandinâ (Tyc
et al. 2017).
Secondary metabolites and hydrolytic enzymes
produced by Bacillus
Secondary metabolites and hydrolytic enzymes are impor-
tant bioactive molecules produced by Bacillus species.
Various studies have evaluated and identified them to be
antifungal, antibacterial and bactericidal but their nemati-
cidal activities are not yet fully known. Although a num-
ber of studies speculate that the nematicidal activity of
Bacillus spp. can be attributed the production of sec-
ondary metabolites (Table 1) and/or hydrolytic enzymes
(Table 2), few investigations have fully characterized these
metabolites or enzymes, and their exact identities and
classifications remain unknown. To our knowledge, not
all of the metabolites in Fig. 1 have been investigated for
their possible nematicidal activity against RKNs of agri-
cultural importance. This emphasizes the need to evaluate
these different secondary metabolite classes for potential
nematicidal compounds.
Cyclic lipopeptides
Species within the Bacillus genera have a wide range of
secondary metabolite gene clusters that encode enzymes,
such as nonribosomal peptide synthetases and polyketide
synthetases, for the production of a variety of lipopep-
tides and PKs (Aleti et al. 2015). These bacteria are the
major producers of cyclic lipopeptides (Rangarajan and
Clarke 2016), which can be classified into four families:
surfactin, iturin, fengycin (Frikha-Gargouri et al. 2017)
and kurstakin (Hathout et al. 2000). Lipopeptides contain

Table 1 Studies in which the nematicidal activity of Bacillus spp. were attributed to secondary metabolite production

Bacillus species Secondary metabolite Plant-parasitic nematode Reference

B. subtilis Bbv57 (KF718836)
B. amyloliquefaciens D1
B. cereus (KUB-15), B. coagulans
(KUB-20), B. cereus (KUB-27)
B. megaterium
B. firmus
B. firmus YBf-10
Surfactin and iturin Meloidogyne incognita (southern root-knot nematode) Ramyabharathi et al. (2018)
Cyclo(D-Pro-L-Leu) M. incognita (southern root-knot nematode) Jamal et al. (2017); Ortiz and
Sansinenea (2017)
Not specified M. javanica (sugarcane eelworm) Abbasi et al. (2017)
Not specified M. graminicola (rice root-knot nematode) Padgham and Sikora (2007)
Not specified M. incognita (southern root-knot nematode), Mendoza et al. (2008)
Radopholus similis (burrowing nematode),
Ditylenchus dipsaci (stem and bulb nematode)
Not specified M. incognita (southern root-knot nematode) Xiong et al. (2015)

330 Journal of Applied Microbiology 127, 326--343 © 2019 The Society for Applied Microbiology
I. Horak et al.
Table 2 Studies in which Bacillus hydrolytic enzymes showed nematicidal activity against Meloidogyne species
Bacillus species Hydrolytic enzyme
B. firmus DS-1 Serine protease
B. pumilus L1 Protease and chitinase
B. megaterium Protease
Bacillus spp. (including Protease
B. cereus and B. pumilus)
B. megaterium and B. circulans Protease, gelatinase and chitinase
B. weihenstephanensis FB25M Collagenase, chitinase,
lipase and protease
a lipid tail and linear or cyclic oligopeptide (Tyc et al.
2017) and are considered amphiphilic molecules since
they exhibit both lipophilic and hydrophilic properties
(P
erez-Garc
ıa et al. 2011; Rangarajan and Clarke 2016).
During the synthesis of lipopeptides, many lipopeptide
isoforms are produced (Frikha-Gargouri et al. 2017).
These isoforms can have different biological activities due
to amino acid residue variation, peptide cyclization and
the traits of the fatty acid chain (nature, length and
branching) (Frikha-Gargouri et al. 2017). Although all
the lipopeptide families have distinct characteristics and
biological activities, when acting synergistically these
lipopeptides can enhance each other’s activities (Aleti
et al. 2015). Lipopeptides exhibit two properties, namely
tolerance and stability, which make them highly suitable
for application in agricultural crop protection practices as
biocontrol agents or biofertilizers (Mongkolthanaruk
2012). They are tolerant to enzymatic breakdown by pro-
nase and proteinase K, and they remain stable at high
temperatures and under low pH conditions. Lipopeptides
directly antagonize phytopathogens by (i) inducing host
plant immunization through root colonization in the rhi-
zosphere and by (ii) interrupting the pathogen’s abilities
to permeate cell membranes (Mongkolthanaruk 2012).
Surfactins are the most comprehensively studied
lipopeptide family and contain approximately 20 different
lipopeptides (Wang et al. 2015). Although surfactins dif-
fer in the composition of their amino acids, they all have
a cyclic heptapeptide structure and they are known to
exhibit various biological activities such as antibacterial,
antiviral and even haemolytic activity (Aleti et al. 2015).
Based on their lipid portion homology and the amino
acid residues located terminally, surfactins can be sepa-
rated into three types, surfactin A, B and C (Wang et al.
2015). They are distinctly different based on the amino
acid present at the position where the lactone ring is
formed. Surfactin A contains ʟ-leucine, surfactin B con-
tains ʟ-valine, and surfactin C contains ʟ-isoleucine
(Wang et al. 2015). Interestingly, surfactin production is
Journal of Applied Microbiology 127, 326--343 © 2019 The Society for Applied Microbiology
Bionematicides from Bacillus
Root-knot nematode Reference
Meloidogyne incognita (southern Geng et al. (2016)
root-knot nematode)
M. arenaria (peanut root-knot nematode) Lee and Kim (2016)
M. graminicola (rice root-knot nematode) Padgham and Sikora (2007)
M. javanica (sugarcane eelworm) Ramezani Moghaddam
et al. (2014)
M. incognita (southern root-knot nematode) El-Hadad et al. (2010)
M. ethiopica (tropical and temperate root- Aballay et al. (2017)
knot nematode)
not necessarily identical for different bacterial strains.
This was demonstrated by Paraszkiewicz et al. (2017)
who found that different strains of B. subtilis exhibited
different surfactin production efficiencies.
The second Bacillus cyclic lipopeptide of agricultural
importance is iturin. A distinct difference between sur-
factin and iturin is that the latter has a cyclic closure of
the lipopeptide structure by a beta-amino group of the
fatty acid (Aleti et al. 2015). Other lipopeptides belonging
to the iturin family and produced by Bacillus species
include bacillomycin D (Gong et al. 2014; Gu et al.
2017), bacillomycin DC (Jin et al. 2017) and mycosub-
tilin (Deravel et al. 2014). Fengycin’s structure contains a
cyclic lactone ring, comprised of a fatty acid chain linked
to a decapeptide. Fengycin has two variants, namely fen-
gycin A and fengycin B (Rangarajan and Clarke 2016).
The former has the amino acid ᴅ-alanine at position 6,
while the latter has ᴅ-valine (Rangarajan and Clarke
2016; Zhao and Kuipers 2016).
A fourth family of cyclic lipopeptides, called kursta-
kins, was discovered almost two decades ago by Hathout
et al. (2000). These lipoheptapeptides are produced by
Bacillus thuringiensis subsp. kurstaki. Kurstakins are asso-
ciated with bacterial cells and spores and can thus not be
retrieved from the bacterial supernatant (B
echet et al.
2012). This is an important consideration when investi-
gating the biocontrol potential of kurstakin. In recent
years, there has been an increasing amount of kurstakin-
based literature being published. B
echet et al. (2012)
reviewed the biosynthesis, structure and properties of
kurstakins, while G
elis-Jeanvoine et al. (2017) recently
reported on the krs locus responsible for encoding nonri-
bosomal peptide synthetases that synthesize kurstakin.
Polyketides
Polyketides are a large class of secondary metabolites
(Mojid Mondol et al. 2013; Palazzini et al. 2016) synthe-
sized nonribosomally (Frikha-Gargouri et al. 2017) by
331
Bionematicides from Bacillus
enzymes known as PKs synthetases (Aleti et al. 2015).
They are structurally diverse (Mojid Mondol et al. 2013)
and exhibit pharmacological properties. PKs produced by
Bacillus species include bacillaene, difficidin and macro-
lactins (Hamdache et al. 2011; Aleti et al. 2015; Palazzini
et al. 2016). Bacillaene is a polyene antibiotic inhibiting
protein synthesis of prokaryotes, but not eukaryotic
organisms. It has a linear structure consisting of two
amide bonds (Hamdache et al. 2011). Difficidin is a
macrocyclic polyene lactone phosphate ester that inhibits
the synthesis of proteins, and macrolactins have a macro-
lide-like structure (Hamdache et al. 2011). A recent study
by Ben Abdallah et al. (2018) investigated the biocontrol
efficacy of B. amyloliquefaciens subsp. plantarum strain
32a and found that this strain not only harboured genes
for the production of macrolactin, bacillaene and diffi-
cidin, but also produced hydrolytic enzymes in vitro.
Through their antimicrobial activity and the activation of
plant defences, PKs are critical in the suppression of plant
disease (Aleti et al. 2015).
Bacteriocins
Bacteriocins are ribosomally synthesized antimicrobial
peptides (Ayed et al. 2015; Berini et al. 2018) produced
by every major bacterial group with over 99% of all bac-
teria producing at least one bacteriocin (Tyc et al. 2017).
Bacillus sp. produce a variety of bacteriocins including
subtilin, ericin, coagulin, sublancin, mersacidin, halodu-
racin, thurincin, cerein, megacin, subtilosin A and liche-
nin (Abriouel et al. 2011). One of the most
comprehensively studied bacteriocins is nisin, produced
by the lactic acid bacteria Lactococcus lactis (Lee and Kim
2011) and used as a food preservative (McAuliffe et al.
2001). When compared to bacteriocins produced by lactic
acid bacteria, Bacillus bacteriocins show greater potential
for important applications such as those in agriculture
(Abriouel et al. 2011). This is evidenced by B. amylolique-
faciens producing the bacteriocin plantazolicin which has
shown to be nematicidal (Liu et al. 2013; Chowdhury
et al. 2015). Bacteriocins act as defence mechanisms
against competing micro-organisms (Berini et al. 2018),
and they are nonlethal to the strains producing the
metabolite, but lethal to other related and unrelated
micro-organisms (Balciunas et al. 2013; Ayed et al. 2015).
This can be attributed to the fact that Bacillus bacteri-
ocins are distinctly diverse in their inhibition abilities
(Ayed et al. 2015) based on their chemical structure,
thermostability, molecular mass, enzymatic sensitivity,
mode of action and modified amino acid presence (Sumi
et al. 2015). These proteinaceous antimicrobial substances
can be classified as either class I, II or III (Balciunas et al.
2013). Lantibiotics are classified as class I bacteriocins
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I. Horak et al.
(Arguelles Arias et al. 2013) and are only a few amino
acid residues in length (Balciunas et al. 2013). Class II
bacteriocins are small peptides that do not contain lan-
thionine (McAuliffe et al. 2001) and can be divided into
five subclasses (class IIa, b, c, d and e). Lastly, class III
bacteriocins are high molecular weight peptides
(>30 kDa) which can lose their structure and become
inactive when exposed to heat (McAuliffe et al. 2001; Lee
and Kim 2011). In a review by Abriouel et al. (2011), the
structure, characterization, classification and environmen-
tal applications (such as biological control) of Bacillus
bacteriocins were comprehensively discussed.
Class I bacteriocins or lantibiotics (McAuliffe et al.
2001) can be viewed as lanthionine-containing (Fuchs
et al. 2011) antimicrobial peptides produced by a variety
of bacterial species (Abriouel et al. 2011). The name is
derived from ‘lanthionine-containing antibiotic’ (McAu-
liffe et al. 2001). Based on their structure and charge at a
neutral pH, they can be divided into two subgroups:
Type A lantibiotics and Type B lantibiotics (Arguelles
Arias et al. 2013; Wang et al. 2015). The former is char-
acterized by a linear secondary structure, positive charge
and a molecular weight of less than 5 kDa, and includes
subtilin and ericin, while the latter is globular and slightly
negative or noncharged such as mersacidin (Arguelles
Arias et al. 2013; Wang et al. 2015). Lantibiotics are ini-
tially synthesized as precursor peptides, but undergo
post-translational modification to give rise to their char-
acteristic lanthionine or methyllanthionine thioether
bridges (Abriouel et al. 2011). They are thermostable
(Arguelles Arias et al. 2013), and notable Bacillus lantibi-
otics include subtilin, amylolysin, subtilosin and mer-
sacidin. Subtilin is a lantibiotic exhibiting bactericidal
activity and whose structure is characterized by five unu-
sual amino acids namely lanthionine, b-methyllanthio-
nine, D-alanine, dehydroalanine and dehydrobutyrine
(Abriouel et al. 2011). Subtilin is produced by B. subtilis
as an adaptive response to environmental changes, such
as decreased nutrient availability or starvation. Higher
levels of subtilin are produced during nutrient depriva-
tion, as opposed to when sufficient amounts of nutrients
are available (Abriouel et al. 2011). Amylolysin, on the
other hand, has the ability to inhibit the biosynthesis of
cell walls, which is accomplished by interacting with the
peptidoglycan monomer carrier, lipid II (Sumi et al.
2015).
Hydrolytic enzymes
A number of hydrolytic enzymes are produced by Bacillus
such as lipase (Sharma et al. 2018), protease (Lee and
Kim 2016) and chitinase (Chandrasekaran et al. 2014;
Zhang et al. 2014), and several studies have reported on
I. Horak et al.
the nematicidal activity of these enzymes (Table 2).
Chitin is the second most abundant natural biopolymer
on earth and serves as an energy source when it is
degraded by bacterial chitinase (Hamid et al. 2013).
Chitinase can be categorized as either endochitinases or
exochitinases based on their mode of action (Berini et al.
2018) and can further be classified into five classes and
two families, including family 18 and 19 of glycosyl
hydrolases (Hamid et al. 2013; Subbanna et al. 2018).
The majority of bacterial chitinases belong to family 18
(Hamid et al. 2013). Due to their nematicidal properties,
chitinases are attractive for use in biological control of
pests. Their biological activity is attributed to their capac-
ity to degrade chitin by breaking the glycosidic bonds
within the biopolymer. This is highly advantageous in
biocontrol as chitin is a major component of the most
vital structures of pests, such as the peritrophic matrix of
insects and eggshells of nematodes (Berini et al. 2018).
The use of chitinases in the biocontrol of nematodes is
especially promising as the eggs are a vulnerable life-stage
of nematodes (Verdejo-Lucas et al. 2002). Thus by
degrading eggshells, the nematode juveniles will be
exposed to environmental conditions and possible preda-
tion or infection by other antagonists. Moreover, chiti-
nases are harmless to nontarget organisms due to the
absence of chitin in plants and vertebrates (Berini et al.
2018). Subbanna et al. (2018) recently reviewed the pro-
spect of using chitinolytic bacteria such as Pseudomonas
and Bacillus, in agricultural pest management discussing
their potential against insect pests, plant pathogenic fungi
and PPNs. Ben Abdallah et al. (2018) determined the
chitinase activity of B. amyloliquefaciens subsp. plantarum
strain 32a by cultivating the bacteria on colloidal chitin
agar plates. Digestion of the colloidal chitin was indica-
tive of chitinase production (Ben Abdallah et al. 2018).
Other Bacillus species exhibiting chitinase activity include
B. pumilus (Rishad et al. 2017), B. subtilis (Chan-
drasekaran et al. 2012), B. thuringiensis, Bacillus licheni-
formis (Gomaa 2012), B. cereus (Liang et al. 2014; Seo
et al. 2014) and B. safensis (Pandya and Saraf 2015). Of
these species, B. subtilis (Abbasi et al. 2014) and B. pumi-
lus (Lee and Kim 2016) are antagonistic towards RKNs.
It is however important to note that the production of
microbial chitinase is regulated by a receptor-inducer sys-
tem. This means that chitinase production can be signifi-
cantly influenced by cultivation conditions and media
constituents (Cheba et al. 2018).
Intracellular metabolites, extracellular metabolites
and the metabolic overflow theory
Intracellular metabolites are enclosed by cell structures
such as the cell membrane or envelope, acting as a
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Bionematicides from Bacillus
mechanical barrier (Pinu et al. 2017), and collectively,
these metabolites are referred to as the endometabolome
(Mashego et al. 2007). Little is known about the secretion
mechanisms of primary intracellular metabolites, but the
release of intracellular metabolites into the extracellular
medium has been explained by the metabolic overflow
theory (Pinu et al. 2018). The loss of bacterial metabo-
lites to the external environment cannot be avoided
(Pande and Kost 2017), and the exometabolome refers to
all of these metabolites excreted outside of the cell.
The metabolic overflow theory states that intracellular
intermediates of metabolic pathways can be secreted
extracellularly when these intermediates accumulate
within the cell (Granucci et al. 2015) due to imbalanced
intracellular metabolic pathways (Pinu et al. 2018). In
response to environmental conditions, metabolite secre-
tion can be seen as a fundamental biochemical function
which reflects the internal metabolic state of microbial
cells. Yet, it has been observed that metabolites which do
not accumulate within the cell are directly excreted into
the extracellular medium in response to environmental
signals (Pinu et al. 2018).
Even though the secretion of many metabolites can tra-
ditionally be explained by the metabolic overflow theory, a
time-series metabolomics study by Granucci et al. (2015)
demonstrated that the theory was not explanatory for the
secretion of many metabolites. Samples were taken at three
distinct intervals during cultivation in a bioreactor—at the
anaerobic steady state, during the transition between
anaerobic and aerobic steady states, and at the aerobic
steady state, and the intracellular and extracellular metabo-
lites of Saccharomyces cerevisiae were measured in parallel
to one another. Results indicated that the intracellular and
extracellular levels of aspartate, 2-oxoglutarate, benzoate
and decanoate changed in opposite directions, while a
total of six metabolites were uniquely detected in the spent
culture media (Granucci et al. 2015).
Pinu et al. (2018) used times-series metabolomics data
from different micro-organisms (bacteria and yeast) to
confirm seven possible relationship patterns between the
levels of intracellular and extracellular metabolites.
i Both the intracellular and extracellular levels of the
metabolite increase (classical metabolic overflow the-
ory).
ii Both the intracellular and extracellular levels of the
metabolite decrease.
iii The intracellular metabolite levels increase, while the
extracellular metabolite levels decrease (classical
metabolite uptake).
iv The intracellular metabolite levels decrease, while the
extracellular metabolite levels increase (metabolite
efflux).
333
Bionematicides from Bacillus
v The intracellular metabolite levels increase, without
any significant change in the extracellular metabolite
levels.
vi The intracellular metabolite levels show no significant
change, but the extracellular metabolite levels vary
significantly (either increasing or decreasing).
vii The metabolite is produced within the cell, but is
only detected extracellularly.
This last relationship pattern is the most perplexing as
the extracellular metabolites can either increase or
decrease over a period of time without being detected
intracellularly. The suggestion of species-specific or
metabolite-specific secretion patterns are not yet possible
(Pinu et al. 2018). The presence of extracellular metabo-
lites is the result of the metabolites originally being part
of the media composition, metabolite secretion by cells,
cell lysis, or polymer degradation. When compared to
intracellular metabolites, extracellular metabolites have
lower turnover rates due to the dilution, low abundance
or complete absence of enzymes outside the cell (Pinu
et al. 2017). According to Granucci et al. (2015), the
analysis of extracellular metabolites is technically advanta-
geous over that of intracellular metabolites most likely
due to their accessibility and ease of handling (Villas-
B^oas et al. 2007). When the focus of a study is on the
extracellular metabolites, the metabolites are quantified in
the supernatant when the microbial cells are separated
from the cultivation media (Pinu et al. 2017) by means
of filtration or centrifugation (Mashego et al. 2007; Gran-
ucci et al. 2015). The supernatant obtained is then used
for the metabolomics analysis (Pinu et al. 2017) while the
cell pellet is discarded. The extracellular metabolite profile
obtained from such metabolomics analysis is informative
with regards to the metabolic state of cells cultivated in a
specific medium under certain environmental conditions
(Granucci et al. 2015).
Optimum cultivation conditions for metabolite
production
A number of studies have investigated the influence of cul-
tivation conditions on Bacillus spore production (Posada-
Uribe et al. 2015; Xu Zhou et al. 2017), while others
focused on conditions for optimum protease production
(Ray et al. 2012; Sarker et al. 2013). It was found that alka-
line media and elevated temperatures reduced the volume
of spores, while aeration and agitation rates affected spore
cell density (Posada-Uribe et al. 2015; Xu Zhou et al.
2017). Maximum protease production for both B. subtilis
and B. licheniformis was reached at 40°C with an optimum
pH of 60–65 (Ray et al. 2012). However, few studies have
investigated the influence of culture conditions on
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I. Horak et al.
specifically metabolite production. The metabolic status of
micro-organisms is culture condition-sensitive (Shen et al.
2016). Consequently, the use of microbial cells for metabo-
lomics can be problematic during analysis since cells
require cultivation in growth media and specific media
components could artificially increase metabolite levels
(Marcinowska et al. 2011). The production of metabolites
by micro-organisms is influenced by various environmen-
tal factors (Kim and Kim 2017) such as the cultivation
media used (complex media vs a minimal media), cultiva-
tion method (batch culture vs continuous culture), tem-
perature, the period of incubation, pH, light and moisture
(Tyc et al. 2017), and it is important that these environ-
mental parameters be kept stable (Shen et al. 2016). For
this reason, it is imperative that the cultivation conditions
for metabolomics studies are optimized to ensure that the
maximum amounts of significant metabolites are produced
to ensure detectability.
Culture media
A variety of cultivation media has been used in microbial
metabolomics studies, including Luria-Bertani broth
(Marcinowska et al. 2011; Joghee and Jayaraman 2014),
M9 (Joghee and Jayaraman 2014), minimal broth, nutri-
ent broth (Kouremenos et al. 2014) and universal 13C-
labelled medium (Zhong et al. 2018). The cultivation
media used have a direct influence on the type and
amount of metabolites produced during incubation, due
to differences in nutrient content and the resulting meta-
bolic pathways. However, few studies have investigated
the effect of different types of cultivation media on
microbial metabolic activities. Recently, a metabolomics
study by Kim and Kim (2017) examined the effect of cul-
tivation media (minimal media vs complex media) on
the metabolite profiles obtained for Escherichia coli and S.
cerevisiae. The complex media used were Luria–Bertani
broth and YP broth, while M9 media and a yeast nitro-
gen base broth were used as the minimal media for
E. coli and S. cerevisiae, respectively. Cultivation in the
complex media resulted in higher microbial growth rates
and final cell densities, in comparison with minimal
media. Furthermore, it was found that cultivation in the
complex media yielded significantly higher levels of
amino acids, while the production of sugars, fatty acids
and sugar alcohols was higher in the minimal broth (Kim
and Kim 2017). Medium- and organism-specific intracel-
lular metabolites were compared to determine whether
the metabolite profile obtained depended on the type of
cultivation medium or on the micro-organism itself. Sim-
ilar media exhibited similar metabolite profiles, irrespec-
tive of the micro-organism (E. coli or S. cerevisiae), and it
was concluded that media-specific differences outweighed
I. Horak et al.
organism-specific differences showing that uptake and
metabolism of nutrients is a critical factor in determining
metabolite production (Kim and Kim 2017).
Mosquera et al. (2014) aimed to maximize biomass
production and the biological activity of the biocontrol
agent B. subtilis EA-CB0015 by optimizing the culture
media. This optimized media, containing 334 g l1 of
glucose and 325 g l1 of yeast extract, resulted in
increased cell densities and biological activity with higher
fengycin production (Mosquera et al. 2014). A study by
Ayed et al. (2015) aimed to isolate and characterize a
bacteriocin-like peptide from B. amyloliquefaciens An6.
The effect of two important variables, incubation period
and nitrogen and carbon sources, on the production of
bacteriocins was also investigated. After comparing four
different media (including Luria–Bertani broth), the cul-
tivation conditions were optimized and it was found that
an incubation period of 48 h resulted in maximal bacteri-
ocin production. When compared to incubation at 30°C,
a similar growth curve was observed than when B. amy-
loliquefaciens An6 was incubated at 37°C. However,
antimicrobial activity was higher at 30°C than at 37°C.
Ultimately, the optimized culture media contained
20 g l1 of starch and 10 g l1 of yeast extract (M2), had
an initial pH of 80 at 30°C, and was incubated at
200 rev min1 (continuous agitation; Ayed et al. 2015).
Cultivation media not only affect the production of
soluble metabolites, but also that of volatiles. The pro-
duction of different VOCs due to different media con-
stituents was demonstrated by Ratiu et al. (2017). When
E. coli was cultivated in three different growth media
(Mueller Hinton, trypticase soy broth and a glucose-
enriched minimal salts media), different classes of vola-
tiles were produced due to different metabolic pathway
activity when different components were available for uti-
lization.
Batch culture vs continuous culture
The cultivation method used can either impede or facili-
tate the production of certain enzymes and metabolites of
interest. For example, chitinases can be produced using
liquid batch, fed batch, continuous or solid-state fermen-
tations (Berini et al. 2018). According to Shen et al.
(2016), the most frequently used cultivation methods for
metabolite studies are batch culture and chemostat (con-
tinuous). The former entails cultivating cells in a fixed
volume of growth media and thus a fixed amount of sub-
strate (Maier 2009) under specific laboratory conditions
(temperature, pH, nutrient availability, pressure, etc.)
which are maintained for the entire incubation period.
Nutrients are not renewed, and as a result of the continu-
ously changing culture environment, growth, substrate
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Bionematicides from Bacillus
utilization and the production of metabolites will also
change. Different growth phases thus exist in batch cul-
ture. On the other hand, when chemostat is used, fresh
media (and thus a fresh supply of nutrients) are continu-
ously added while spent culture media are continuously
removed. This method of cultivation occurs in a bioreac-
tor consisting of various components such as a feed
reservoir, pump, fermenter, stirrer and weir (Shen et al.
2016). Jin et al. (2015) compared the production of
iturin A by B. subtilis cultivated by means of a two-stage
glucose-feeding strategy (fed batch), as opposed to a
batch culture. The aim of the study was to optimize
iturin A production due to its potential application as a
biopesticide. With this novel feeding strategy, a twofold
increase in iturin A production was observed when com-
pared to that of the batch culture. After 110 h, a maxi-
mum iturin A concentration of 112 g l1 was reached. It
was concluded that this approach is not only suitable for
the production of iturin A, but could be used to enhance
the production of other secondary metabolites produced
by B. subtilis.
Incubation period and bacterial growth phase
As previously noted, metabolites are characterized by infi-
nite half-lives and are thus taken up, produced, degraded
or excreted (Villas-B^ oas et al. 2007). However during
metabolic activities, metabolites are not only formed, but
also converted into different compounds. This underlines
the importance of the incubation period which could
have a direct influence on the metabolites or compounds
detected. If the bacterial cultures are incubated for too
long a period, some metabolites of possible significance
could already have been converted into other com-
pounds. Contrary to this, if the incubation period is not
long enough, metabolites of possible significance may not
have been synthesized yet (e.g. secondary metabolites
which are produced during the stationary phase of
growth). This occurrence demonstrates the importance of
constructing bacterial growth curves prior to cultivation.
It will enable the determination of the appropriate period
of cultivation for the production of biologically active
molecules such as secondary metabolites.
The growth phase at which bacteria are sampled for
metabolomics analysis is a another important considera-
tion since different types of metabolites are produced
during the four phases (lag phase, exponential growth
phase, stationary phase and decline phase; Favre et al.
2017). This is important as each phase is associated with
certain physiological changes within the microbial cell.
Yet many studies fail to elaborate on how the secondary
metabolites were distinguished from the primary metabo-
lites and often make assumptions based on the growth
335
Bionematicides from Bacillus
phase used for sampling. During the lag phase, which is
dependent on inoculum size and culture media used, the
cells must first adapt (Maier 2009). This is followed by a
phase of exponential growth. Although cells still grow
and divide during stationary phase, there is no net
growth and this phase is usually brought on by one of
two events: depletion or accumulation. When the carbon
and energy sources of an important nutrient are depleted,
the bacterial cells have no more substrate left to utilize
for growth and survival; this results in stationary phase.
Alternatively when bacterial waste products start to accu-
mulate, this will also inhibit growth and lead to station-
ary phase. During this phase, endogenous metabolism—
growth on dead, lysed cells—is often observed. Lastly,
when there is a net loss of viable cells, the death phase
has been reached (Maier 2009). Unfortunately, few stud-
ies have investigated what differences are observed in
microbial metabolomes during their growth phases (Favre
et al. 2017) and this should be addressed in future
studies.
In a quest for the detection of species-specific metabo-
lites produced by four marine bacteria (Persicivirga
mediterranea TC4 and TC7, Pseudoalteromonas lipolytica
TC8, and Shewanella sp. TC11), Favre et al. (2017) inves-
tigated the influence of cultivation parameters on the
metabolic activities of these micro-organisms. The media
used for cultivation, the phase of growth and mode of
culture were all assessed. The growth curves obtained
from measuring the optical densities (OD600) demon-
strated that the bacterial cultures grew differently and
that the duration of the exponential growth phase was
not the same for all four strains. Moreover, the number
of metabolites detected in the growth phases also differed
as more Shewanella sp. TC11 metabolites were detected
during the exponential phase while more were detected
for P. lipolytica during the stationary phase. Although the
majority of m/z signals were detected in both the expo-
nential and stationary growth phase, higher amounts
were detected during stationary phase for more than two-
thirds of the signals (Favre et al. 2017). Recently, Yun
et al. (2018) demonstrated that growing yeast in cultiva-
tion media with different carbon sources resulted in
growth phases being reached after different periods of
time. When the yeast Yarrowia lipolytica was cultivated in
complex media containing glucose or glycerol, or mini-
mal media containing glucose, stationary phase was
reached after a period of 30 h. However, when cultivated
in minimal media containing glycerol, the stationary
phase was only reached after 43 h. During exponential
phase, the metabolite profiles of Y. lipolytica grown in
complex media were grouped together in the PCA model
but during stationary phase the metabolite profiles for
both the complex and minimal media were separate.
336 Journal of Applied Microbiology 127, 326--343 © 2019 The Society for Applied Microbiology
I. Horak et al.
Although yeast was used, this study raises some impor-
tant questions about media and carbon selection as well
as the chosen growth phase for sampling. All of which
are key considerations when cultivating bacteria for the
purpose of metabolomics studies.
The importance of the growth phase was also high-
lighted in a metabolomics-based study by Jin et al.
(2013) who aimed to determine what intracellular meta-
bolic changes occur during different growth phases of
Halomonas sp. KM-1, a poly(3-hydroxybutyrate) (PHB)
producing bacterium. It was concluded that phase-speci-
fic mechanisms are involved and that metabolite changes
are related to the accumulation of PHB during different
phases of growth. The specific growth rate of the bacteria
also influences the expression of Bacillus enzymes such as
the protease subtilisin. According to Seok Oh et al.
(2002), a specific growth rate of 035 h1 is considered
optimal for maximum subtilisin expression and longest
period of subtilisin production. During metabolomics
studies, it is essential to construct bacterial growth curves
prior to cultivation to determine the appropriate period
of cultivation for the production of biologically active
molecules, such as secondary metabolites. Growth curves
can easily be constructed by measuring cell mass with a
spectrophotometer in terms of absorbance (optical den-
sity). The cell growth process should be strictly controlled
to ensure that the data obtained are representative and
reproducible (Marcinowska et al. 2011).
Temperature, salinity, pH and oxygen availability
The temperature at which bacterial cultures are incubated
is another important consideration for optimum metabo-
lite or hydrolytic enzyme production. When investigating
the effect of fermentation conditions and nitrogen
sources on the production of chitinase by Bacillus sp. R2,
Cheba et al. (2018) found that incubation at 30°C
resulted in the highest observed chitinase activity. Culti-
vation of B. cereus 8A at 30°C was also found to more
appropriate for the production of the bacteriocin cerein
8A as opposed to cultivation at 25 or 37°C with the latter
resulting in decreased bacteriocin activity (Bizani and
Brandelli 2004). Ayed et al. (2015) also stated that there
is often a correlation between growth temperature and
bacteriocin production. Salinity, pH and oxygen availabil-
ity (aerobic vs anaerobic cultivation conditions) can also
affect metabolite production. Important cellular pro-
cesses, such as primary and secondary metabolite biosyn-
thesis regulation, can be affected by fluctuations in the
initial pH of the media (Ayed et al. 2015). The produc-
tion of surfactin depends on pH, while oxygen influ-
ences the expression on mycosubtilin (Mongkolthanaruk
2012). Joghee and Jayaraman (2014) investigated the
I. Horak et al.
relationship between salt-stress and metabolic changes of
four bacterial halophiles including Bacillus aquimaris
VITP4. The bacteria were cultivated in minimal media
(M9) and a complex media (LB), and it was found that
under the same salt-stress, the bacteria had a higher
growth rate when cultivated in the complex media
(Joghee and Jayaraman 2014).
Although some studies have aimed to optimize culture
conditions for the maximum production of bioactive
molecules and biological activity (Angelo et al. 2015), it
is important to bear in mind that replicating the diverse
and fluctuating environmental conditions that promote
metabolite production in laboratory investigations is
quite challenging (Tyc et al. 2017). Since the production
of biologically active metabolites is especially influenced
by the amount of nutrients available for uptake and use,
variation often observed in the biocontrol efficacy of field
applications could likely be attributed to nutrition
(Anderson and Kim 2018).
Using metabolomics to detect possible
nematicidal compounds
Various studies have found Bacillus secondary metabolites
to be responsible for the observed antagonism towards
plant pathogens such as Meloidogyne (Mendoza et al.
2008; Xiong et al. 2015; Ramyabharathi et al. 2018).
However, such studies are often speculative or fail to
elaborate on how the secondary metabolites were detected
or distinguished from primary metabolites. Consequently,
the identities of these metabolite features remain
unknown. A ‘metabolite feature’ is a molecule with a
unique mass to charge ratio (m/z), which will be an accu-
rate indication of mass and retention time in chromatog-
raphy. Identifying a feature does not always mean it has
been identified to the compound level. In this context,
the identification of a feature means that the feature was
identified as a discriminatory feature explaining the sta-
tistical differences between metabolite profiles from dif-
ferent Bacillus species. Metabolomics, defined as the
comprehensive analysis of all the metabolites present
within an organism (Villas-B^ oas et al. 2007; Carnicer
et al. 2016; Patejko et al. 2017), can therefore be used to
detect and qualitatively and quantitatively characterize
discriminatory metabolite features with nematicidal
potential.
Traditionally, the production of secondary metabolites
was detected by means of phenotypic tests. However,
there has been rapid progress in high-throughput tech-
nologies and platforms to analyse global mRNA, pro-
teins and metabolite profiles of cells (Horgan and
Kenny 2011; Meng et al. 2014). Aliferis and Chrysayi-
Tokousbalides (2011) described metabolomics as a
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Bionematicides from Bacillus
valuable high-throughput screening tool for the discov-
ery of biologically active substances that are highly selec-
tive and show unique mechanisms of action. Therefore,
modern approaches such as metabolomics could provide
new insights into the identity of ‘metabolite features’
which are responsible for the observed nematicidal activ-
ity of some bacterial strains.
Metabolomics studies can either be targeted or untar-
geted. Targeted metabolomics refers to a method in
which a pre-identified list of metabolites are measured
(Dudley et al. 2010) making it possible to search for a
specific set of metabolite features in bacterial metabo-
lomes. It is highly precise, accurate and hypothesis-
driven (Alonso et al. 2015) with the extraction of
metabolites based on the chemical properties of the
metabolite classes of interest (Patejko et al. 2017).
Therefore, if scientists have a pre-identified list of possi-
ble nematicidal compounds produced by different Bacil-
lus species (such as a shortlist of specific secondary
metabolites or hydrolytic enzymes), a targeted metabolo-
mics approach could be followed to determine whether
the species of interest produces one or more of the
compounds. On the other hand with an untargeted
approach, all of the metabolites present in a sample are
comprehensively analysed (Roberts et al. 2012) in order
to detect the maximum amount of metabolites (Gao
and Xu 2015). This includes analysis of metabolites with
unknown chemical functional groups (Roberts et al.
2012). Although time intensive (Patti et al. 2012), untar-
geted metabolomics is useful when comparing entire
metabolite profiles of several bacterial strains of which
some exhibit nematicidal activity. Statistically significant
differences between features observed in the metabolite
profiles of nematicidal bacterial strains compared to
those without nematicidal activity might explain the
observed activity.
Conclusion and future prospects
The development of biological nematode control prod-
ucts based on bacterial secondary metabolites and hydro-
lytic enzymes is somewhat difficult. Most research
conclusions only assume that nematicidal activity
observed by rhizobacterial filtrates can be attributed to
secondary metabolites and/or hydrolytic enzymes. Yet in
many cases, they fail to identify the nematicidal features
in question. From an experimental perspective, this
emphasizes the need to reach consensus in the literature
about the differentiation between primary and secondary
metabolites and how to best cultivate bacterial strains for
optimum metabolite and enzyme production. This is
imperative as the metabolic status of micro-organisms
directly depends on cultivation parameters such as the
337
Bionematicides from Bacillus
media and initial pH, method (batch vs continuous cul-
ture), incubation period and temperature.
For the development of nematicidal metabolites or
enzymes, media selection is a very important considera-
tion. The chosen media should provide sufficient nutri-
ents to the micro-organism to ensure rapid growth but
also promote the production of metabolites and enzymes
of interest. Moreover, the media constituents should not
interfere with the metabolomics analysis or artificially
increase metabolite levels. In theory, the use of a continu-
ous culture for metabolite production is considered more
productive than a batch culture system. However, some
argue that the longer duration of a continuous system
increases the probability of culture contamination. Ulti-
mately, the cultivation method chosen will strongly influ-
ence the product produced. Furthermore, the appropriate
incubation period should be determined through the
construction of bacterial growth curves which will also
aid in determining the best period of time to sample (e.g.
stationary phase for secondary metabolites). The tempera-
ture and pH utilized will also depend on the type of
bacteria and metabolites or enzymes of interest.
Based on the literature reviewed in this article, several
important questions are posed by the authors: (i) Are the
nematicidal compounds produced by Bacillus species
always secondary metabolites and/or hydrolytic enzymes,
or could they be primary metabolites or even other com-
pounds? (ii) What cultivation medium (complex vs mini-
mal medium) would be more suitable for the cultivation
of nematicidal Bacillus species to ensure optimum
metabolite and/or hydrolytic enzyme production? (iii)
What differences or similarities will be observed in the
metabolite profiles of Bacillus species cultivated in contin-
uous culture as opposed to batch culture? (iv) Do media-
specific differences always outweigh organism-specific dif-
ferences in bacterial metabolite and/or hydrolytic enzyme
production? And (v) are the nematicidal metabolites pro-
duced by Bacillus species intracellular or extracellular
metabolites?
Although each study is unique in terms of the aims
and objectives, studies aimed at characterizing nematici-
dal compounds should first clear up the basic aspects and
address posed questions. To further bionematicide devel-
opment, a more detailed and integrated research
approach, taking all possible variables in the production
of bioactive molecules into account, should be followed.
Acknowledgements
The financial support of the National Research Founda-
tion (NRF) South Africa and the German Academic
Exchange Service (DAAD), as a bursary (grant numbers
111891 and 106783), are hereby acknowledged. Opinions
338 Journal of Applied Microbiology 127, 326--343 © 2019 The Society for Applied Microbiology
I. Horak et al.
expressed and conclusions arrived at are those of the
authors and are not necessarily to be attributed to the
NRF or DAAD.
Conflict of Interest
The authors declare no conflict of interest.
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