Review Article - (2021) Volume 12, Issue 8
The Intriguing Extrapolations of Haemolysis Assay as Screening Criterion for Selecting Biosurfactant- Producing Microorganisms in Petroleum Industries Process-Conditions
and Falode OA1,3
Adenike AOO, Centre for Petroleum, Energy Economics and Law, University of Ibadan,
+ 08130049657, Email:
Author info »
Commonly-used screening techniques for determination of biosurfactants production by microorganisms include
haemolysis assay, generally depicted to confirm the ability of microorganisms in production of biosurfactants.
Diameters of zones of haemolysis surrounding microbial colonies are considered as quantitative indication of
biosurfactant production whereas; haemolytic reactions on blood agar plates are specifically associated with
pathologic types of erythrocyte lysis by microorganisms, due to haemolysins production. Haemolytic microorganisms
can destroy erythrocyte membranes, by compromise in integrity of cytoplasmic membranes, through pore-forming
mechanisms, multiple-hit mechanism, formation of sphaerocytes, derangement of membrane integrity, detergentlike
action, or lipase activity. Relative levels of acute toxicity, cell invasiveness and virulence factors, which can
make biosurfactants become opportunistic pathogens that use haemin or haemoglobin as a source of iron, have
also been reported. Haemolysins are further classically defined as exotoxins that can be thermostable, and can
cross membranes of microorganisms. Haemolysis assay thus, identifies haemolytic microbial strains with lytic,
pathogenic, toxigenic, and/or virulent potentials, rather than biosurfactant-producing potential, as the assay
does not correlate particularly with specific characteristics of biosurfactants’ production. However, based on new
insights and perspectives appropriately extrapolated for the first time in this report, microbial haemolysis assay is
considered, the easiest, most-economical, non-animal-based, highly-determinative, reliable and sensitive biosafety
selection criterion protocol, for selection of safe and environmental-friendly biosurfactant candidates, for the
petroleum industries’ process conditions.
Biosurfactants; Blood agar; Microbial haemolysis assay; Microbial toxicity; Petroleum industries
Microbial surfactants (biosurfactants) are one of the wide ranges
of extracellular compounds that are produced by microorganisms,
particularly, bacteria and fungi (yeasts, moulds and mushroom),
more especially when grown on hydrophobic substrates. They
are surface-active microbial amphiphilic compounds, which are
produced on living surfaces, mostly, on microbial cell surfaces or
excreted as extracellular hydrophobic and hydrophilic moieties.
These characteristics thus, confer on the biosurfactants-producing
microorganisms, the ability to accumulate between fluid phases,
and also possess the characteristic property for reducing surface and interfacial tension, at surfaces and interfaces respectively. By
accumulating at the interfaces of immiscible fluids, biosurfactants
have been reported to be able to increase the solubility,
bioavailability and subsequent biodegradation of hydrophobic or
insoluble organic compounds [1-10]. However, biosurfactants use
similar mechanisms to the chemical surfactants but mostly with
certain more established advantages [11-13].
In addition to having many unique properties and applications, the
ability to exhibit biosensoactives (surface‐ active) properties, which
lower the surface tension and the interfacial tension of their growth
media, allow biosurfactants to play diverse key beneficial roles [13-28]. Furthermore, the vast structural diversities that characterise biosurfactants, may also explain the reason for their continual
intrigue to scientific interests [16,29,30]. Increased environmental
awareness has been the main driver for the search of biosurfactants,
as replacement for chemical surfactants [2,13,24,29-34].
Benefits and applications of biosurfactants in petroleum
Biosurfactant production is considered one of the key technologies
for development in the 21st century, and biosurfactants are widely
applicable in almost every area of human endeavours, especially
in the field of petroleum technology and processes. Specifically,
due to their efficacy as dispersion and remediation agents,
biosurfactants have several potential applications across the oilprocessing
chains, and in the formulations of petrochemical
products, microbial enhanced oil recovery, anti-corrosives; biocides
for sulphate-reducing bacteria; emulsification and emulsified fuels;
de-emulsification; oil waste treatment; enhancement of crude
oil transportation through pipelines; crude oil spill clean-ups/
bioremediation of crude-oil polluted soils; including the removal of
crude oil from contaminated soils and water bodies by indigenous
microbes, biodegradation. Some other benefits of biosurfactants
are environmental remediation processes like- oil storage bottom
sludge tank cleaning; sediment remediation, soil washing and soil
flushing, extraction of bitumen from tar sands, and extraction of
hydrocarbon compounds from oil shales, in order to utilise them
as a substitute for petroleum energy fuel [10,12,16,17,22,24,35-86].
Biosurfactants being diverse amphiphilic molecules,
with wide structural and functional diversities,
and because great diversity also exists a m o n g
biosurfactant roducing microorganisms, there is adoption of
different screening techniques for their determination; although,
almost all the screening methods can give qualitative and/or
quantitative results [87-93].
Some screening methods for biosurfactant production, which are
basically automated and/or miniaturised rapid-screening assays,
are available in present times. However, t h e
major regular direct and indirect biosurfactants screening
assays are presented in Figure 1 [19,88,58,90,93,94-122].
Figure 1: Common direct and indirect non-automated biosurfactants
Screening assays for selecting biosurfactant-producing
Each biosurfactant screening method, as presented in Figure 1, has
its advantages and disadvantages; so, a combination of different
methods has been suggested as appropriate for successful screening
of biosurfactants [15,87,88,121,123]. In addition to the physiological
nature of the biosurfactant-producing microorganisms, screening
for biosurfactant producers somehow depends, both on the type
of carbon source(s) present, and also the types and amounts of
other nutrients in the screening media [92,124-130]. The screening
medium used will therefore, tremendously influence production or
non-production of biosurfactants; and also influence the type and
amount of biosurfactants produced .
Haemolysis assay in biosurfactant determination
It was reported that haemolytic activity of biosurfactants was first
discovered when Bernheimer and Avigad  recorded that
surfactin, the biosurfactant produced by B. subtilis, lysed erythrocytes.
Afterwards, haemolysis assay for biosurfactant determination was
also reportedly developed by Mulligan et al. . Following the
development of haemolysis assay for biosurfactant determination,
Carrillo et al.  also claimed to have discovered an association
between haemolytic activity and surfactant production. Several
studies similarly reported the impossibility of biosurfactant
production without haemolytic activity, as haemolysis has been
referred to as a determination of biosurfactant [87,106,113,121,131-134]. Whereas, as summed up by Kabir et al. , very few
bacteria would have the selective advantage of lysing erythrocytes,
yet haemolysis test has always been considered an ideal assay for
determining surfactant production, as it is commonly claimed
that biosurfactants cause lysis of erythrocytes, and this is usually
the principle adopted in the haemolysis assay for biosurfacts
In several studies on biosurfactants, haemolysis assay on blood agar
plates has always been an exclusive experimental screening method
to monitor biosurfactant production . Based on the reference
that biosurfactant-producing capacity in liquid medium was
found to be associated with haemolytic activity, the use of blood
agar lysis (haemolysis assay) was considered and recommended
as appearing to be a good primary (and in few cases, secondary)
screening criterion/method for biosurfactant production, by
surfactant-producing microbial strains, and regarded as indicative of
biosurfactant production [16,19,25,87,88,96,102,106,113,117,135-145].
Preparation of blood agar for haemolysis assay
Blood agar is an enriched and differential solid growth medium
with general composition of-blood agar peptone: 10 g/l; yeast
extract: 3 g/l; NaCl: 5 g/l; blood: 100 ml/l (as the basal medium),
of which specified mls. of human or animal (rabbit, sheep, horse
or cattle) blood is added, for the growth of many microorganisms,
especially, the fastidious microorganisms (i.e., microbial species
that do not grow easily on general purpose culture media, which
are microbial culture media that lack special nutrients). Firstly,
for haemolysis assay, the isolated microbial colonies may be subcultured
from primary plates, by four-corner streaking or repeated
microbial colony transfers (for mostly fungal isolates) on appropriate
sterile culture media, in order to obtain pure microbial cultures.
The pure microbial cultures can then be inoculated on any of the
various modified blood agar plates, such as, Zobell marine medium
supplemented with 5% fresh human blood . Other basal culture media to which human or animal blood can be added, in
order to prepare blood agar include, blood agar base, tryptone soy
agar, nutrient agar, plate count agar, Mueller-Hinton agar, potato
dextrose agar, Sabouraud dextrose agar, etc.
Inoculation of blood agar plates is usually followed by incubation
at 25-37°C for 24-72°C or 96 hrs, depending on the bacterial or
fungal species. The blood agar plates are then visually inspected
for haemolysis (clear zone) around the haemolytic microbial
colonies. Secondly, the initial isolation of suspected biosurfactantproducers
may also primarily be done on blood agar plates,
based on the acclaimed ability of many biosurfactants to lyse
erythrocytes, which then results in haemolysis around suspected
biosurfactant-producing microbial colonies, on the blood agar
Blood agar and haemolysis
Haemolytic microbial strains cause lysis of erythrocytes, and exhibit
haemolytic zones, which can be complete or partial haemolysis
around the haemolytic microbial colonies. As introduced by Brown,
the three basic types of haemolysis (haemolytic reactions) that can
be observed on blood agar plates are designated, alpha (∝), beta (β)
and gamma (γ) haemolysis [148,149], as denoted in Figure 2.
Figure 2: Haemolytic reactions on blood agar plates.
Alpha-haemolysis (α-haemolysis) is a greenish discoloration that
surrounds a haemolytic microbial colony, growing on blood agar
plate. This type of haemolysis represents a partial (greenish) lysis
or incomplete decomposition (reduction) of the haemoglobin of
the erythrocytes (red blood cells). Alpha haemolysis is caused by
hydrogen-peroxide produced by alpha-haemolytic bacteria or fungi,
which oxidise haemoglobin to green methaemoglobin, in the
medium surrounding the colony. Thus, alpha-haemolytic microbes
thus, produce greenish diffusible appearance on blood agar plates
Beta-haemolysis (β-haemolysis) represents a complete haemolysis
(complete breakdown) of the haemoglobin of the red blood cells
surrounding a microbial colony, on blood agar plate, giving a
transparent or translucent clearing of the blood agar around the
microbial colony. Beta haemolysis is more pronounced when
the blood agar plate is incubated anaerobically, although some
microorganisms are weakly beta-haemolytic species .
Gamma-haemolysis (γ-haemolysis/non-haemolysis) is the third type
of haemolytic reaction, in which there is actually no haemolysis at all,
as there is lack of haemolysis in the area surrounding the microbial
colony on blood agar plates. Gamma-haemolysis show neither
typical alpha nor beta haemolysis, due to no haemolytic change
around the microbial growth on blood agar plates (http://www.encyclopedia.com/science/encyclopedias-almanacs-transcriptsand-maps/blood-agar-hemolysis-and-hemolytic-reactions). There
may however, be, slight brownish discolouration (not haemolysis)
on the blood agar plates . Zonee of alpha and beta-haemolysis
surrounding microbial colonies on blood-agar plates are however,
designated as hallmark phenotypic features of various pathogenic
Haemolysins as microbial toxins and virulence factors
Haemolysins, sometimes classified as enzymes, are lipids and
proteins that have been extensively reported and studied in bacteria,
fungi, various species of plants, invertebrates, mammals, and also denoted as perforins, in fungi, plants, invertebrates, and mammals
[153-168]. Haemolysins cause lysis (destruction) of erythrocytes
(red blood cells), by destroying their cell membrane (Figure 3), with
release of their haemoglobins; thereby, providing iron, for bacterial
growth. Through haemolysins enzymatic attack on phospholipids,
the cell membranes are subsequently destabilised , as shown
in Figure 3.
Figure 3: Permeability of bacterial cell membrane by haemolysin
Pore formations in microbial cell membranes, derangement of
microbial cell membrane integrity, detergent action, or lipase
activity are the major mechanisms by which microbial haemolysisns
cause haemolysis [69-173]. It was further proposed that the
hydrophilic part (the cationic part) of biosurfactants initiate
electrostatic interaction with the negatively charged components
of the bacterial cell membranes; while the hydrophobic portion
was supposed to permit the peptides to insert into, and permeate
the bacterial cell membranes . Some haemolysins however,
attack the phospholipid of the host cytoplasmic membrane, by
using phospholipases lecithinases, and the phospholipids, lecithin
(phosphatidylcholine), often used as substrate; although, some
haemolysins affect the sterols of the host cytoplasmic membrane .
In addition to bacterial growth, due to the release of haemoglobins
after red blood lysis; thereby, providing iron, pathogenicity; are
also reported, and the responsible haemolysisns considered as
toxins. So, being identified as extracellular toxic proteins that
are produced by several microbial species, all of which possess
a certain pathogenic potential; haemolysins have usually been
further considered as virulence factors , and sublytic effects
of haemolysin can alter host cell regulation and lead to cell death
[176,177]. Due to production of cytolytic toxins, haemolysins from
several bacterial and fungal strains have been confirmed to possess
lytic activities that correlate with severity of haemolysin–induced
infections, sometimes, with high mortality rates [168,177-180].
Haemolysis is also considered a pathogenicity indicator tool [180-182], and haemolysins have similarly been linked to increased
severity of infections, and concretely associated with virulence, in
addition to pathogenesis or pathogenicity [152,183-188].
Among the well-known diverse toxigenic microbial haemolysins
are, small β-pore-forming toxins, alpha-haemolysin monomers
secreted by Staphylococcus aureus, aerolysin, secreted by Aeromonas
hydrophila; α-toxins, secreted by Staphylococcus aureus and Clostridium
septicum; cholesterol-dependent cytolysins (CDCs), like streptolysin
O, secreted by Streptococcus pyogenes, and listeriolysin O, secreted
by Listeria monocytogenes or AB toxins, like the diphtheria toxin,
secreted by Corynebacterium diphtheriae, as well as the toxic fungal
haemolysins like, nigerlysin, aerolysin, ostreolysin, pleurotolysin A
and B etc. [166,169,189-193].
Mechanisms of pore-forming toxins are depicted in Figure 4;
thereby, it is designated that pore-forming toxins like thermostable
direct haemolysin (TDH) are also known to induce haemolysis,
by incorporating into cell membranes to form pores .
Pore-forming toxins are secreted by microbial pathogens in a
water-soluble form that binds to the target cell, then generally
multimerises into an amphipathic structure that finally inserts into
the target cell membrane, and then forms a pore . The native
thermostable direct haemolysin (TDHn) is transformed into nontoxic
fibrils, rich in beta-strands, by incubation at 600°C, to form
the incubated thermostable direct haemolysin (TDHi). The TDHi
fibrils are dissociated into unfolded states by further heating above
800°C (TDHu) but the protein is trapped in the TDHi structure
by slow cooling of TDHu, while rapid cooling of TDHu results in
refolding of the protein into toxic TDHn .
Figure 4: Pore-forming toxins mechanisms.
Haemolysins lyse erythrocytes, which results in the release of
iron, an important growth factor for microorganisms, especially
in pathogenicity, and during infections [196,197], as it is certain
that numerous pathogenic microorganisms grow in the host by
using haemin or haemoglobin as a source of iron [198-201]. Several
fungal haemolysins have also thus, been proposed as virulence
factors [202-204]. In addition to cell adherence, cytotoxicity and cell
invasiveness, haemolysis also has an additional clinical significance,
in being regarded as a virulence factor [205,206]. Furthermore,
microbial haemolysins promote opportunistic infections and other
clinical conditions, and also presented as risk factors in hospitals
patients [202,207-209]. The expression of a haemolytic protein, with capabilities to lyse erythrocytes, has also been suggested as
providing survival strategy for fungi during opportunistic infections
. The haemolysin, which enabled the fungus to disrupt blood
cells, contained negatively charged domains that could also be
detected in infected patients [166,211-213].
Research studies have shown that another application of fungal
haemolysins has been their use as biomarkers for personal exposure
to fungi or species-specific identification of opportunistic fungal
diseases [166,214,215-217]. There is therefore, considerable interest
in the development of diagnostic assays for detecting haemolysins
as biomarkers of allergic and disseminated fungal exposure .
In actual fact, fungal haemolysins have been useful as biomarkers
for exposure to indoor fungi because they can be measured in
bodily fluids and environmental samples .
Extrapolations of the haemolysis screening assays in the
determination of biosurfactants
According to Mulligan et al.  and Walter et al. , the
technique of using blood agar plate haemolysis assay to screen for
biosurfactant production on soluble substrates was shown to be
quick and reliable. Some authors also believed that haemolysis
screening method can be used to limit the number of samples, when
selecting biosurfactant-producing microorganisms. In some cases,
further screening for biosurfactant-producing microorganisms is
only carried out, after screening for positive haemolytic activity
. The clear zone of haemolysis around the microbial colony on
blood agar plates has commonly been related to the ability of the
microbes to produce surfactants, while the diameter of the clear
zone usually considered as a qualitative indicator of biosurfactant
production [96,218]. It has however, been reported that haemolysis
assay is not a specific method for biosurfactant production, since
not all biosurfactants have haemolytic activities, basically due to
presence of compounds other than biosurfactants [102,113]. Such
other compounds include, virulence factors, toxins, and other lytic
enzymes that can lyse erythrocytes . It was also reported that
biosurfactants that are poorly diffusible may not lyse erythrocytes
nor cause haemolysis [220,221]. Furthermore, in some studies,
haemolysis assay was found to exclude many good biosurfactantproducers,
while in some reports, microbial strains with positive
haemolytic activity were found to be negative for biosurfactant
production . There were a number of reports as well, which
confirmed that microorganisms that were positive as biosurfactantproducing
with the use other selection criteria, were negative for
biosurfactant production when screened for haemolytic activity
The poor specificity of haemolysis screening assay had also been
confirmed, in that, it can give a lot of false-negative and falsepositive
results [113,117]. In addition, it was reported that the
diffusion restriction of surfactant can inhibit the formation of
clearing zones on blood agar plates. Likewise, over-incubation of
the blood agar plates may cause microbial overgrowth, which can
lead to accumulation of microbial waste-products that may lyse
the blood on blood agar plates; thereby, giving false appearance
of biosurfactants, which are actually not present (http://www.encyclopedia.com/science/encyclopedias-almanacs-transcriptsand-maps/blood-agar-hemolysis-and-hemolytic-reactions). Until
now, haemolytic microbial strains were generally believed to be biosurfactant-producers. Whereas, from the microbiological, clinical, pathophysiological and public health points of
interpretations, the degree by which erythrocytes are haemolysed
on blood-based culture media, is basically used to distinguish
haemolytic and non-haemolytic microorganisms. Moreover,
visualising the physical appearance of haemolysis on cultured
blood agar plates has been used as a tool to determine the aetiologic
(disease-causing) microbial species of various microbial infections
In more recent times, biosurfactants have been generally
considered as biodegradable, non-toxic (or minimally toxic), and
eco-friendly/environmental-friendly compounds that are released
by microorganisms [40,54,55,61,66,223-225]. But, apparently,
most of the biosurfactants proposed in literature are reportedly
produced by pathogenic microbes involved in pathogenesis,
while relative levels of acute toxicity have also been recorded
among significant numbers of surfactant-producing bacteria and
fungi. The pathogenicity associated with haemolysis is therefore,
a cause for concern, considering it being a commonly adopted
biosurfactant potential and /or biosurfactant screening criterion.
Therefore, contrary to the reports that biosurfactants-producing
microorganisms used in some studies were generally recognised as
safe (GRAS) , most of the microorganisms referred to as GRAS
may still harbour one or more pathogenic/toxigenic/virulence
factor(s), which can make them opportunistic pathogens .
Many well-characterised biosurfactant producers have been
confirmed as pathogenic microbial species [24,203,227].
Conversely, haemolysis and haemolysins are specifically indicative
of pathogenic and/or virulent or/and toxigenic status, rather
than biosurfactant-production. The haemolytic action of certain
bacteria and fungi on blood agar is so striking that haemolysis has
been classified as very significant in clinical diagnosis of microbial
importance. Due to the pathogenicity of some biosurfactantsproducing
microorganisms [24,203,227,228], they were therefore,
more recently, considered not appropriate for scaled- up production
. The detection of virulence genes coding for haemolysis and the
determination of the antimicrobial resistance, in addition to the
factors that contribute to pathogenicity and toxicity can contribute
to better understanding of the need for better selection criteria
of biosurfactants-producing microorganisms, and applications of
their products , in the petroleum industries.
As earlier reported, literature on the production and analytical
detection of biosurfactants is overwhelming, with assertions of
high yields, and with mostly over-exaggerated estimates, due to the
use of flawed or inaccurate analytical techniques . However,
none of the previous documented assertions on haemolysis assay,
in biosurfactant determinations or contrariwise, highlighted
the vivid microbiological and safety implications of haemolysis
assay in biosurfactants-producing microorganisms. Based on the
tremendous afore-mentioned intriguing justifications, haemolysis
assay more appropriately identifies microbial strains with
haemolytic (pathogenic/toxigenic/virulent) potentials. It can then
be extrapolated that haemolysis assay (i.e., lyses of erythrocytes)
on blood agar plates are more of diagnostic or determinative
tools for microbial pathogenicity, rather than biosurfactants
productions, and can therefore, not be conclusively confirmatory of
biosurfactant-production, nor considered an appropriate selection
criterion for biosurfactants. Thus, the likely or real pathogenicity,
and/or virulence and toxicity of biosurfactants-producing microbes
need to be appropriately assessed by haemolysis assay, prior to their potential applications in various petroleum industries, more
especially, as they may be multi-antimicrobial resistant haemolytic.
From the petroleum industries perspectives, polycyclic aromatic
hydrocarbons (PAHs) and naphthenic acids (NAs) are well-known
to be toxic contaminants of environmental concern . It is
therefore, of necessity to ensure that additional hazardous concerns
associated with petroleum activities are not introduced into the
environment. A variety of microbial taxa are able to synthesize
biosurfactants but it is ideal to isolate biosurfactants-producing
microorganisms from appropriate and safe sources. From the ideal
petroleum microbiology, public health, and hydrocarbon-processing
points of view, the microbial strain profile and the ecological niche
matter, as they determine the physiological status and metabolites
production of the putative microorganisms. Therefore, it is
proper to isolate biosurfactants-producing microorganisms from
same or closely related ecological nich(es), for same physiological
characteristics, extended survival, and maximal production of
biosurfactant metabolites. Furthermore, toxic agents can cross
microbial membranes , into the hosts; so, haemolysis assay, is
hereby, suggested as, a highly determinative and qualitative screening
assay indicative of the biosafety potentials, for the determination
of pathogenic, toxigenic and/or virulent biosurfactant-producing
microorganisms in the petroleum industries.
Biosurfactants are highly important microbial compounds of
tremendous benefits but their significant public health concerns,
especially regarding their haemolytic potentials serving as
biosurfactant property are presently misconstrued. Bacterial and
fungal haemolysins have been used as diagnostic tools, and/or
biomarkers but microbial toxicity is undesirable in selected microbial
candidates for various beneficial activities, such as, biosurfactantsproductions.
Based on the intriguing afore-listed justifiable reasons,
it can be noted and extrapolated that haemolysis assay; using blood
agar is not so reliable, sensitive or suitable for determination of
biosurfactant production, as it does not correlate particularly with
specific characteristics of biosurfactants’ production. However,
haemolysis assay is quite appropriately as, a reliable and sensitive
safety bioassay, in routine monitoring, for pathogenic/toxigenic,
and virulence determinations and regulations, as well as for
selecting safe and environmental-friendly biosurfactant-producing
This work was supported by the Klinkinberg Engineering Solutions
Nigeria Limited [Grant number KESNL 0001, 2019].
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and Falode OA1,3
Centre for Petroleum, Energy Economics and Law, University of Ibadan, Oyo State, Nigeria
Department of Microbiology, Faculty of Science, University of Ibadan, Oyo State, Nigeria
Department of Petroleum Engineering, Faculty of Technology, University of Ibadan, Oyo State, Nigeria
Pegasus-Zion Community and Environmental Health, Nigeria
Citation: Adenike AOO, Falode OA (2021) The Intriguing Extrapolations of Haemolysis Assay as Screening Criterion for Selecting Biosurfactant- Producing Microorganisms in Petroleum Industries Process-Conditions. J Pet Environ Biotechnol. 8: 431.
Jul 10, 2021 / Accepted Date:
Sep 18, 2021 / Published Date:
Sep 21, 2021
Copyright: © 2021 Adenike AOO, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.