Critical Reviews in Biotechnology

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Screening concepts, characterization and

structural analysis of microbial-derived bioactive
lipopeptides: a review

Piotr Biniarz, Marcin Łukaszewicz & Tomasz Janek

To cite this article: Piotr Biniarz, Marcin Łukaszewicz & Tomasz Janek (2017) Screening
concepts, characterization and structural analysis of microbial-derived bioactive lipopeptides: a
review, Critical Reviews in Biotechnology, 37:3, 393-410, DOI: 10.3109/07388551.2016.1163324

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CRITICAL REVIEWS IN BIOTECHNOLOGY, 2017
VOL. 37, NO. 3, 393–410
http://dx.doi.org/10.3109/07388551.2016.1163324

REVIEW ARTICLE

Screening concepts, characterization and structural analysis of

microbial-derived bioactive lipopeptides: a review
Piotr Biniarza, Marcin Łukaszewicza and Tomasz Janeka,b
a
Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland; bDepartment of Inorganic Chemistry, Faculty of Pharmacy, Wroclaw
Medical University, Wroclaw, Poland

ABSTRACT ARTICLE HISTORY

Lipopeptide biosurfactants are surface active biomolecules that are produced by a variety of Received 1 April 2015
microorganisms. Microbial lipopeptides have gained the interest of microbiologists, chemists and Revised 21 December 2015
biochemists for their high biodiversity as well as efficient action, low toxicity and good biodegrad- Accepted 23 December 2015
ability in comparison to synthetic counterparts. In this report, we review methods for the produc-
KEYWORDS
tion, isolation and screening, purification and structural characterization of microbial lipopeptides. Biosurfactants; chemical
Several techniques are currently available for each step, and we describe the most commonly uti- analysis; lipopeptides;
lized and recently developed techniques in this review. Investigations on lipopeptide biosurfac- microbe; purification;
tants in natural products require efficient isolation techniques for the characterization and screening
evaluation of chemical and biological properties. A combination of chromatographic and spectro-
scopic techniques offer opportunities for a better characterization of lipopeptide structures, which
in turn can lead to the application of lipopeptides in food, pharmaceutical, cosmetics, agricultural
and bioremediation industries.

Introduction
Microorganisms produce a wide variety of chemical com-
pounds. Some of these molecules are already exploited
in different fields, such as medicine and industry, while
others are likely waiting to be discovered and developed.
Among the thousands of microbial-derived compounds,
biosurfactants (BS) are one such emerging class of mole-
cules.[1] BS are amphiphilic secondary metabolites that
have surface and interfacial activity as every molecule
contains both hydrophobic and hydrophilic moieties.
Thus, BS are capable of reducing the surface tension or
solubilizing hydrophobic substances in water. They are
considered to be ‘‘green’’ alternatives for chemical surfac-
tants that are widely used in different aspects of human
activity, such as industry, farming and medicine.[1]
BS are produced mainly by bacteria (e.g. Bacillus and
Pseudomonas genera) and yeasts, such as Candida or
Yarrowia [2–5] BS are a broad group of molecules that
include lipopeptides (LP), lipoproteins, glycopeptides,
phospholipids, etc. LP are comprised of a hydrophobic
tail, which is usually a fatty acid, linked to a hydrophilic
head between 4 and 12 amino acids. The hydrophilic
head may be linear or contain a lactone ring as is found
in cyclic LP. The best known cyclic LP are surfactins, itur-
ins, fengycins, lichenysins, viscosins, amphisins and puti-
solvins. Due to their chemical structure and biological
and physiochemical properties, LPs are a particularly
promising class of BS.[2,3,6,7]
LPs exhibit activities that make these molecules
potentially useful in industry, environmental protection
and medical fields. Previous research has highlighted
their potential use as bioemulsifiers, heavy metal-bind-
ing compounds, antibiotics, antifungals and antitumor
agents. Potential applications of LP/BS has been the
subject of a number of thorough reviews and research
publications.[2,3,8–12]
The broad spectrum of properties attributed to LP can
be credited to their modularity, which results in a variety
of possible structures. A change in a single amino acid
within the molecule can cause an activity modifica-
tion.[13,14] LPs are synthesized by macromolecular pro-
tein complexes known as non-ribosomal peptide
synthetases (NRPS). The modular organization of these
complexes and the possibility of rearrangement within
the NRPS genetic clusters enables the production of a
great variety of LP. The molecular basis of LP

CONTACT Tomasz Janek tomasz.janek@umed.wroc.pl Department of Inorganic Chemistry, Faculty of Pharmacy, Wroclaw Medical University, Borowska
211a, Wroclaw 50-552, Poland
Supplemental data for this article can be accessed here.
ß 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/Licenses/by-nc-
nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or
built upon in any way.
394 P. BINIARZ ET AL.
biosynthesis has been extensively reviewed by many
authors.[7,14,15]
While some research teams have focused on the pro-
duction, purification and characterization of already
known LP/BS,[16–19] others seek for innovative applica-
tions for BS.[8,11,20,21] The third branch of BS research
is searching for novel molecules.[22–25]
The identification of novel LP is critical to enrich our
knowledge of BS and will provide researchers with mol-
ecules that may have desirable characteristics, such as
high temperature stability, activity in a broad range of
pH and salinity levels, satisfactory biodegradability and/
or new biological activities.[2,26–28]
Although many valuable papers on BS have been
published, we found no concise review that introduced
and described a workflow of LP/BS research. The goal
here is to outline the techniques that, in our opinion,
should be followed in a comprehensive study on novel
LP/BS, beginning from the isolation and screening of
BS-producing microbes, through the production and
purification of the novel compounds and finally, com-
pound characterization. It is hoped that this review on
LP can assist researchers whose studies are dedicated to
LP but can also be applied to other classes of BS due to
their similar physicochemical properties.
Isolation and screening of BS-producing
microorganisms
There are a variety of traditional, popular and recently
developed methods for the isolation and screening of
microbial BS producers. Some of these methods have
been exhaustively tested and reviewed while others are
rarely employed.[22,24,29,30] Thus, these techniques
will not be discussed and instead, we will focus mainly
on novel high-throughput methods.
The workflow of BS research begins with the isolation
of microorganisms followed by screening and identifica-
tion of BS-producing strains (Figure 1). These microbes
(e.g. bacteria, archaea and fungi) are isolated from envir-
onmental samples, such as soil and water, collected
from around the world.[23,29,32,33] A small percentage
of environmental isolates normally produce BS.[33]
Interestingly, this number can rise to 25% in samples
from hydrocarbon or heavy metals-polluted sites or
even more dramatically (up to 80%) when enrichment
cultures with hydrocarbons are used.[24,29,32–34,]
All methods adopted for the screening of BS pro-
ducers share an important limitation – only microbes
susceptible for artificial cultivation can be isolated and
analyzed. Moreover, the conditions and media used for
preliminary cultivation of the isolates can influence the
metabolism of microorganisms and inhibit production of
BS. Thus, it is likely that the distribution of BS producers
in different environments is underestimated.[33]
According to our current knowledge, no metagenomic
approach has been widely used for the screening of
BS-producing strains [35–38]. Such an approach could be
designed for LP-producers due to the following: the high
homology of NRPS operons in different species, the num-
ber of already designed primers in the literature and the
possibility of utilizing different databases and platforms,
like BLAST, NRPS predictor or AntiSMASH as exam-
ples.[39–44] In our opinion, this may be the most cost-
effective and efficient way to analyze LP/BS produced by
microorganisms in different environments.
After all, the isolation and cultivation of pure micro-
bial cultures must first be carried out before screening
and/or other analyses.[36] Standardized microbiology
protocols are often used to isolate and culture the
microbes. First, samples are taken from a specific type
of source (e.g., water or soil), diluted and plated on vari-
ous types of agar plates (Figure 1). After a few days of
incubation, single colonies are selected and transferred
to a liquid medium to cultivate individual microbes
separately.[17] As an alternative, enrichment culturing
is a popular modification of this standardized
approach.[33,45] In this technique, environmental sam-
ples are incubated in a liquid medium using hydrocar-
bon as the main carbon source to promote the growth
of hydrocarbon-degrading microbes. Samples taken
from enrichment cultures are subsequently diluted and
agar plated as previously described. The correlation
between microbial growth on hydrophobic carbon sub-
strates and the production of BS has been reported,
although researchers should keep in mind that there are
always exceptions from every rule.[33]
Recently, protocols for high-throughput isolation
approaches have been published, but these techniques
are still in development.[46–48] We were unable to
identify reports that described the utilization of high-
throughput isolation procedures followed by HTS for BS
production; therefore in our opinion, this is a field worth
developing. High-throughput isolation techniques also
have the potential to assist with the cultivation of
currently ‘‘uncultivable’’ BS-producing microbes.[47]
Therefore, these techniques can be useful for estimating
the distribution of BS producers in different niches as
well as uncovering ‘‘unknowns’’ of BS production.
When isolates are grown and single, pure colonies
are isolated, they can be screened for the production of
BS (Figure 1) with several highly utilized, traditional
techniques that involve direct and/or indirect methods
(Table S1). But in a past few years, new screening proto-
cols have appeared. We present the current knowledge
 CRITICAL REVIEWS IN BIOTECHNOLOGY 395

Figure 1. Standardized workflow for the identification and characterization of BS/LP. BS-producing microbes can be found in envir-
onmental samples (A) and isolated with a variety of microbiological techniques (A1, B) to pure colonies (C). Next, microorganisms
are screened with one or more HTS techniques (e.g. drop collapse assay or its modifications) for the ability to produce BS (D). BS-
positive strains are then identified (e.g. by 16S rDNA sequencing; D1). Production of BS should be additionally confirmed (e.g. by
TLC; E). BS/LP can be produced in standard laboratory Erlenmeyer flask (F). To obtain a pure compound (G) that can be used in dif-
ferent examinations, several isolation/purification techniques can be used. A structure of pseudofactin II (H) is given as an example
of a LP molecule that passed through the presented workflow [10–12,31]

on these newly developed indirect (Table 1) and direct
techniques (Table 2) below.
All types of surfactants can be detected simultan-
eously with indirect screening methods,[22,25,30,51]
while direct methods can usually be used for only one
type at a time.[56,57] Indirect methods can provide pre-
liminary information on BS production kinetics and
yield. Although these methods may result in false posi-
tive or false negative qualitative data as well as under-
or overestimating BS yields. The various indirect techni-
ques also differ in sensitivity, and only some are suitable
for high-throughput screening (HTS). Moreover, these
results do not provide information on the nature and
structure of any detected surface active compound(s).
Indirect screening methods (Table 1 and Table S1) tend
to be used to reveal changes in the surface properties
of microbial cultures (e.g. measuring surface tension
during cultivation) and/or to detect emulsification activ-
ity or antimicrobial/hemolytic activity of culture super-
natants.[26,54] Due to simplicity, indirect screening
techniques are usually used for preliminary screening
for BS producers.[25,29] Specific techniques are
described in Table 1 and Table S1.
While indirect screening techniques are inexpensive
and easy to carry out, they have several disadvantages
(Table 1 and Table S1) and are being replaced by direct
approaches (Table 2). The more sophisticated direct
methods (Table 2) can answer several different ques-
tions. Certain direct techniques can reveal additional
properties, such as structural information. The decision
as to which method would be the most suitable should
be considered with a specific scientific question in
 396

Table 1. Indirect, novel screening methods used for revealing BS-producing microbes. These HTS procedures can be used for detecting all types of surfactants and are mostly based
on measuring changes in the surface properties of examined sample
Application Quantitative
Method name Applicability Short description Simplicity in HTS Precision results Sensitivity Comments References
P. BINIARZ ET AL.

Modified drop-collapse BS Each well of 96-well microplate is þþþ þ þ þ þ One of the most utilized [22,49,50]
coated with thin layer of oil (e.g. min- methods for detection of BS,
eral oil) prior to analysis. Then a 5 ml together with a simple drop
drop of a sample is added to the cen- collapse analysis (cf. Table S1).
ter of a well and observed after
1 min. Presence of surfactants in solu-
tion will result in a larger diameter of
a drop in comparison to water drop.
Microplate meniscus BS 100 ml of microbial culture super- þþþ þþ þ þþ þ Rough quantitative results can [29]
shape assay natant is put into a 96-well micro- be obtained by diluting the
plate and diluted serially. Then, the sample serially on a micro-
plate is investigated – an image of a plate. Applied in a limited
grid is viewed through the plate. number of research works.
When surfactants are present in solu-
tion, the surface of a liquid in well
forms a concave lens which distorts a
view of a grid.
Atomized oil assay BS Microbial cultures are spotted on agar þþþ þ þ þþ þ Designed for uncovering sur- [45,50,51]
plate and grown. BS solution can be face-enhanced production of
also spotted on a plate. Then, a mist BS (yet, many strains produce
of mineral oil is applied on a surface BS only if grown in liquid
of agar with an airbrush. A halo media).
around BS spots or BS-producing
microbes can be observed and
measured.
Polydiacetylene (PDA) Surfactin/ionic PDA vesicles, which act as sensors for þþ þþ þþ þþ þþ Applied in a limited number [52]
vesicles surfactants surfactants, are synthesized from of research works.
PCDA (10,12-pentacosadiynoic acid).
Solutions containing BS and PDA
vesicles are mixed in microplate and
the color change is measured.
Simplicity is an outcome of availability and price of laboratory equipment and materials needed in a certain test as well as demand for skilled staff: very easy (þ þ þ), easy (þ þ), difficult (þ); application in HTS: very
good (þ þ þ), good (þ þ), poor (þ); precision is an outcome of repeatability of a certain test and the probability of receiving false-positive or false-negative results: very precise (þ þ þ), precise (þ þ), imprecise
(þ); quantitative results: very good (þ þ þ), good (þ þ), poor (þ), not applicable (); sensitivity is the relative concentration of surfactant that can be detected: very good (þ þ þ), good (þ þ), poor (þ), not applic-
able ().
Table 2. Direct, HTS and emerging methods designed for the fast and accurate detection of LP or LP-producing microorganisms
Application Quantitative
Method name Applicability Short description Simplicity in HTS Precision results Sensitivity Comments References
NRPS operon detection LP PCR reaction with primers designed þþþ þþ þþ   Detection of PKS (polyketide syn- [39,43,53,54]
with PCR for conserved regions of NRPS is thases) and/or silent NRPS
carried out. Purified microbial DNA operons is possible, what may
or ‘‘colony PCR’’ supernatant can lead to false-positive results.
be both used as a template for
PCR reaction. PCR products are
detected after agarose gel
electrophoresis.
HPLC/UPLC LP/BS Clarified culture supernatant or crude þþ þþþ þþþ þþþ þþ Expensive equipment and certain [17,54,55]
culture extract can be injected on laboratory skills are needed.
RP-HPLC/UPLC column (C18 is Samples should be analyzed
commonly used) and eluted with simultaneously with purified
water/ACN gradient. LP’s are standards for comparison.
detected at a wavelength of
approx. 210 nm. Identification of
eluted compounds can be done by
comparing the retention time or
second derivative of spectrum with
standards.
LC-MS Small molecular Methods are usually similar to HPLC/ þ þþþ þþþ þþþ þþþ Requires use of an expensive and [17,56,57]
mass BS UPLC analysis. The main difference sophisticated laboratory equip-
is the way of detection. Here, dif- ment, which should be oper-
ferent MS approaches/ionizations ated by skilled staff. Studies on
(e.g. ESI) are used and response of the structure of detected com-
MS detector is observed. pounds can be done simultan-
eously by the analysis of MS/
MS spectra.
MS-based approaches LP/BS Several different methods have been þ þþþ þþþ þ þþþ Requires an expensive or even in- [17,58,59]
(potentially) demonstrated e.g. nanoDESI (nano- house built, prototype equip-
spray desorption electrospray ion- ment and skilled staff. Huge
ization), MALDI-TOF (matrix- amounts of data are gener-
assisted laser desorption/ionization ated, what can cause problems
– time of flight) or other. Variety in selection and analysis of
of sample types can be analyzed essential information. Structural
(microbial colonies, microbial cul- data can be obtained
tures or culture supernatants, cul- simultaneously.
ture extracts).
PrISM (proteomic investigation LP Microbes are grown and subsequently þ þþ þþ   Technique is very similar in its [60]
of secondary metabolism) proteome samples are prepared. assumptions to PCR-based
Samples are loaded onto SDS- methods. Due to ‘‘protein-first’’
PAGE gel and high molecular strategy, probability of obtain-
bands (225 kDa) are isolated, ing false-positive results is
digested, and analyzed (LC-MS/MS) reduced (only expressed genes
for unique marker ions of NRPS can be detected). Experience in
and/or PKS enzymes. analysis of proteomic data as
well as expensive laboratory
equipment are essential.
CRITICAL REVIEWS IN BIOTECHNOLOGY

Simplicity is an outcome of availability and price of laboratory equipment and materials needed in a certain test as well as demand for skilled staff: very easy (þ þ þ), easy (þ þ), difficult (þ); application in HTS: very
good (þ þ þ), good (þ þ), poor (þ); precision is an outcome of repeatability of a certain test and the probability of receiving false-positive or false-negative results: very precise (þ þ þ), precise (þ þ), imprecise
(þ); quantitative results: very good (þ þ þ), good (þ þ), poor (þ), not applicable (); sensitivity is the relative concentration of surfactant that can be detected: very good (þ þ þ), good (þ þ), poor (þ), not applic-
397

able ().
398 P. BINIARZ ET AL.
mind. Direct methods developed for recognizing LP
include: PCR-based procedures [43,53,54] and several
extraction methods coupled with analytical techniques
(Table 2). There are also newly-developed, high-through-
put MS-based and proteomics approaches [59,60] that
potentially could replace traditional screening techni-
ques (Table 2). After certain modifications, these techni-
ques can be also adapted for uncovering different
classes of BS.[61]
The screening step is critical for efficient BS research;
thus, screening techniques require careful selection.
Each approach has its own merits and shortcomings
(Table 1, Table 2 and Table S1); therefore, we suggest a
two-part screening procedure as a successful and effi-
cient strategy (Figure 1). In the first step, a HTS tech-
nique would be used to screen a large number of
isolates with relatively high sensitivity and low probabil-
ity of receiving false positive or false negative results.
The perfect HTS approach would provide results that
would also help assess BS activity, yield, or antimicrobial
activity for instance, so only promising candidate strains
will be selected for subsequent analyses. Drop collapse,
various microplate assays, PCR- and MS-based methods
can be used for a large-scale screen of isolates. Since
none of them individually is the perfect technique, other
factors should be considered when choosing the
method, including: researcher experience in microbio-
logical laboratory techniques, available laboratory equip-
ment, the number of isolated microbes to be tested and
the desired level of sensitivity and accuracy (Table 1,
Table 2 and Table S1).
During the second screening step, selected BS-posi-
tive isolates should be tested further to confirm the pro-
duction of BS, identify the specific type of BS produced
and estimate BS yield and/or activity. Measurements of
the surface tension or emulsification activity of clarified
microbial cultures are commonly utilized assays for con-
firming BS production and estimating BS activity and
yield, while extraction of BS followed by TLC is typically
used for receiving general information on the structure
of active compounds (Table S1).[12,25,26,29,30]
These approaches are now replaced by novel techni-
ques, from which different HPLC/UPLC, LC-MS or MS are
probably the most promising, due to high sensitivity,
accuracy and automation. Absorbance (e.g. at 210 nm)
or MS data can be used for detection and quantification
of LP and MS/MS spectra can be adapted for resolving
the structure of active compounds (Table 2). These
methods can be adopted for different types of BS (e.g.
glycolipids).[55,54] Moreover, some of the novel MS-
based procedures (e.g. nanoDESI) have the potential to
be used as universal methods to join the HTS screening
(Figure 1D) and confirmation (Figure 1E) steps with
structural analysis of BS in a single experiment.[59] Such
methods are, in our opinion, the future not only of LP/
BS research but also analyses of different types of active
biomolecules. On the other hand, MS-based procedures
require sophisticated equipment, a highly-skilled staff
and the capability to analyze huge amounts of gener-
ated data (Table 2).
LP research workflow can also begin with an in situ
genome analysis. Whole microbial genomes or their
fragments can be analyzed, or ‘‘mined’’ for the gene
clusters that encode NRPS operons. Until now, there
have only been a few possible ways to detect NRPS
operons in situ (reviewed in. 62,63] The most obvious
method is to compare the nucleic acid sequence of
interest with known sequences using BLAST.[64,65]
However, more accurate tools that are tailored for LP
are also available.[62,63] These tools can not only be
used to detect potential NRPS operons in genomes but
also to predict the possible structure of detected com-
pounds, comparing them with known LP to forecast
activity and to annotate novel compounds.[62,63]
The identification of selected microbes should be car-
ried out at the end of screening (Figure 1) as this know-
ledge can be helpful in following BS-workflow steps
(e.g. in the choice of optimal cultivation medium and
conditions, see next chapter). Traditional API-testing is
time-consuming and expensive, so it will probably be
outdated in the characterization of environmental iso-
lates. 16S rRNA sequencing is currently the most
exploited method for this purpose.[17,32,34] Although
novel, automated and high-throughput MS-based
approaches have emerged recently with the potential
for identifying hundreds of microorganisms in a single
experiment. Different MS techniques can be used to
detect bacterial biomarkers (e.g. proteins, lipids, lipopo-
lysaccharides or low-mass molecules) with measure-
ments accomplished within minutes. Results can be
compared with databases (e.g. Bruker BioTyper or
SARAMIS) to identify the bacteria of interest to the sub-
species level. Although MS-based biotyping procedures
are primarily used in clinical microbiology, they are pre-
dicted to displace biochemical and molecular biology
techniques in microbe identification in the near
future.[66] Isolation and screening for BS-producers
ends when selected strains are frozen and banked in a
laboratory culture collection in 80  C for further culti-
vation and analysis of surface active compounds.
Cultivation of microbes for the production
of LP
Once LP/BS-producing microbes have been isolated and
identified, the efficient production and purification of

surface active compounds is crucial to characterize
structure and function of novel LP/BS (Figure 1) and
require the optimal culture medium and growth condi-
tions for efficient LP production. Optimization of
nutrients and culture condition for the production of
LP/BS is a topic of great interest.[2,16,18,67–72] Here,
we focus on the main parameters that can affect the
efficient production of LP by microorganisms.
LPs are produced by different microbes, including
several Bacillus and Pseudomonas strains [14,17,37] as
well as other genera.[4,17,34] Generally, cultivation pro-
cedures designed for Bacillus and/or Pseudomonas have
been successfully used for the cultivation and produc-
tion of LP by other genera.[17]
Several factors have been previously reported to be
crucial for the biosynthesis of LP. These include: nutrient
composition (carbon and nitrogen sources), fermenta-
tion temperature, pH and culture oxygenation.[4,71]
There are general rules that should be followed for the
efficient production of LP. First, the pH of the culture
should be kept at a neutral level (6.0–7.0), as acidic pH
can inhibit the production of LP or cause their precipita-
tion.[71,73] Second, a correlation between BS produc-
tion and microbial growth has been reported.[67,71,74]
Therefore, nutrients, especially carbon and nitrogen
sources and culture oxygenation should not be limiting
for bacterial growth, although some exceptions have
been reported.[12,75,76] Third, media selection should
be conducted due to reports that rich, complex media,
such as LB (Luria-Bertani) or NB (Nutrient Broth), may
cause decreased LP production.[73,77,78] Fourth,
microbe cultivation at 30  C seems to be optimal for
most LP producers,[58] although some strains prefer
lower temperatures.[75] Finally, microbial culturing
should last long enough for the microorganisms to
reach the stationary phase of growth when the produc-
tion of most BS is maximized.[67,74]
The selection of culture media is an important step in
efficiently generating ample amount of LP/BS for ana-
lysis. Minimal mineral salt media (MSM) are commonly
used for the production of BS.[12,79,80] The compos-
ition of MSM varies in different reports but generally
consists of buffered salts (e.g. K2HPO4, KH2PO4, NaCl,
KCl), a carbon source (e.g. glucose, sucrose, glycerol or
hydrocarbons) and a nitrogen source (e.g. (NH4)2SO4,
NH4NO3, NH4Cl, or KNO3). Increased LP production has
been described in MSM supplemented with trace ele-
ments, such as divalent cations.[73,80] Small amounts of
complex additives, like yeast extract or protein digests,
can also be added to promote bacterial growth and LP
production.[67,81] Simplicity is probably the most
important advantage of MSM as subsequent isolation
steps are more easily performed with higher purity of
CRITICAL REVIEWS IN BIOTECHNOLOGY 399
the final products (see following chapters).[12] However,
BS production in MSM can be relatively low.[71]
The use of complex media has also been investigated
with conflicting reports. Early studies suggested that
BS production decreases when complex media is
used.[73,77,78] However, subsequent studies indicated
that LP production was suitable using a number of dif-
ferent complex media as well as industrial wastes. These
included King’s B medium [50] Landy’s medium,[82]
corn steep liquor,[83] and glycerol waste from biodiesel
production.[27,74]
Optimal medium and cultivation conditions should
be selected for each individual LP/BS producer to reach
maximum productivity, although amounts of LP suffi-
cient for analyses (Figure 1) can also be obtained from
non-optimized cultures.[11,12] Screening for the optimal
medium is usually carried out in standard laboratory
Erlenmeyer flasks by changing one factor (e.g. carbon
source type or concentration) at a time [67].
Oxygenation can be controlled through the adjustment
of agitation speed or by using baffled flasks.[73] This
traditional, time-consuming methodology can be
replaced with newly developed microbioreactors sys-
tems for HTS of optimal media and conditions [76] and/
or mathematical modeling of BS production, such as
Plackett–Burmann screening or response surface
methodology.[18,67,72]
The optimization process for the production of BS
involves the assessment of LP/BS production to allow
the determination of optimal conditions. The production
of LP/BS tends to be frequently overestimated. Thus, it
is important to develop a reliable technique for the
quantification of the molecules in question.[84]
Quantitative techniques employed for LP/BS screening
(e.g. HPLC analysis) can be successfully adapted to pre-
cisely quantify BS (Table 2). However, the use of semi-
quantitative methods in the initial stages of LP research
can usually provide sufficient data. Therefore, simpler
and cheaper methods could be used. Microplate menis-
cus shape or atomized oil HTS assays are worth consid-
ering (Table 1), especially if laboratory equipment access
is an issue.
Isolation and purification of LP
Successful production must be followed by efficient
purification (Figure 1) as pure compounds should be uti-
lized for structural or physical analyses. To obtain pure
samples of the LP from culture broth, efficient isolation
techniques are required.[30] Thus, one important factor
for determining the feasibility of a production process
on a commercial scale is the availability of suitable and
economic recovery and downstream procedures.
400 P. BINIARZ ET AL.

Table 3. Methods for recovering LP from cultivation broths and their advantages
Method type Method name Experimental approach
Analytical-scale Solvent extraction LP dissolves in organic solvents due to
methods the presence of hydrophobic part in
their molecules
Acid precipitation LP become insoluble at low pH values.
Crude BS can be precipitated and/or
centrifuged
Dialysis Difference in solute concentration; water
soluble extracellular compounds are
purified by dialysis; the typical dialysis
session is usually eight or ten hours
overnight at 4  C
Ammonium sulfate precipitation LP are precipitated using (NH4)2SO4
Ion-exchange chromatography Charged LP are eluted from ion-exchange
resins with proper buffer
Solid phase extraction (SPE) The analyte in SPE is eluted in an non-
polar solvent, which disrupts the inter-
action of the analyte and the station-
ary phase
Adsorption on polystyrene resins LP are adsorbed on polymer resins and
subsequently desorbed with organic
solvents
Preparative-scale Membrane ultrafiltration LP form micelles at concentrations above
methods the critical micelle concentration;
therefore, they can be separated from
other molecules in cultivation broth by
sequential ultrafiltration
Adsorption on wood-activated carbon LP are adsorbed on activated carbon
Foam fractionation In this method, foam is collected
(through fractionation column) and
acidified with HCl down to pH 1.0–2.0
to precipitate LP, which can be
extracted with organic solvents
Advantages References
Recovery and partial purification, [12,89]
reusable nature of organic
solvents;
This method is easy, inexpensive [90–92]
and readily available to recover
crude BS such as LP;
Effective in isolation of micelles [93]
biosurfactants, reusability, fast
recovery;
Efficient in recovery and partial [94,95]
purification of crude BS;
High purity, reusability, fast recov- [96]
ery; high-throughput
Fast, one-step recovery, high level [97]
of purity, reusability; high-
throughput
Fast, one-step recovery, high level [88]
of purity, reusability; high-
throughput
There are many merits of ultrafil- [86,98,99]
tration: No need for chemicals
One-step recovery High level
of purity Simple automation;
high-throughput
High level of purity, reusability, [100]
cheap recovery from continu-
ous culture high-throughput
Cheap recovery from continuous [101–103]
culture;

Efficient and high-throughput processing methods are
needed for the maximum recovery of LP/BS.
Conventional and unconventional methods for the
recovery of LP have been reported in recent
years.[85–87]
The main advantage of isolation and purification
methods is their ability to operate in a continuous mode
for recovering LP with high level of purity. A single
downstream processing technique is often not enough
for the recovery and purification of LP/BS. In such cases,
a multi-step recovery strategy using a sequence of con-
centration and purification steps is more effective. With
such a multi-step recovery process, it would be possible
to obtain LP/BS at any required degree of purity.[88]
The most common methods for the purification of
microbial LP include solvent extraction, ammonium sul-
fate precipitation, ultrafiltration and dialysis (Table 3).
One of the simplest methods for the recovery of LP is
acid precipitation.[104] Acid precipitation is carried out
using concentrated HCl to lower the pH to 2.0, which
neutralizes negative charges on LP, making them less
soluble in the aqueous phase. The precipitated LP can
then be collected for analysis. This method is easy, inex-
pensive, and readily available to recover crude LP from
cell-free supernatant. Other more commonly used
methods are foam fractionation, which depends on the
foaming capabilities of LP, adsorption-desorption on
polystyrene resins and ion exchange chromatography.
These procedures take advantage of BS properties, such
as their surface activity or their ability to form micelles
or vesicles, and are particularly applicable for the large-
scale continuous recovery of extracellular BS from cul-
ture broth. In addition, these methods can operate in a
continuous mode for recovering BS with a high level of
purity [105] to produce amounts on a commercial scale.
The most widely used techniques are organic solvent
extractions with chloroform and methanol, petroleum
ether, ethyl acetate, n-hexane and ether.[26,106,107]
Recently, ethyl acetate has been successfully used to
recover LP produced by Pseudomonas [12,108] and
Bacillus.[89] This organic solvent can be used to substan-
tially reduce recovery expenses and minimize environ-
mental hazards.[109]
Identification, characterization and
quantification of LP by various
chromatographic and spectroscopic techniques
There are many chromatographic techniques used to
characterize and quantify molecules, including thin layer

chromatography (TLC), preparative TLC (PTLC), column
chromatography (CC) or modern semi-preparative/pre-
parative HPLC. Each technique has its own strengths
and weaknesses and should be selected based on the
nature of the target compound, such as solubility,
charge, stability and molecular size. Taking these factors
into account, the choice of chromatographic methods
and the stationary phases to be used are important for
designing a purification system.
Other efficient spectroscopic techniques have been
utilized for investigations on LP, such as 1H and 13C
nuclear magnetic resonance NMR; [110] Fourier trans-
form infrared (FT-IR) spectroscopy [30] and MS, to char-
acterize and evaluate chemical and biological
properties.[111] LC-MS has been the most widely
used.[112] These techniques will be reviewed in the fol-
lowing sections.
Thin layer chromatrograpy
TLC separates compounds in a mixture and can be used
to determine the number of components in solutions,
identify these compounds and test their purity.[113] TLC
techniques can be used to detect BS production by sep-
arating cell-free culture supernatants on silica gel plates
and characterizing the chemical nature of BS.[30] TLC
can also initially characterize BS as LP with selective
developing reagents as LP appear as red spots in the
presence of ninhydrin.[114] Furthermore, LP have spe-
cific retention factor (Rf) values for every solvent or solv-
ent mixture. Rf values can be used in compound
identification by comparing the unknown sample with
Rf values of known LP. Several TLC systems are designed
for the analysis of LP (Table S2).
TLC is one of the most versatile techniques for the
identification of natural surface active compounds pro-
duced by microorganisms. It can also be used for moni-
toring the quality and purity of raw BS extracts. Due to
the fact that BS extracts usually occur as a combination
of various types of surface active compounds, their sep-
aration still remains a big challenge, as only pure com-
pounds can be used for the process of identification
and characterization.[58,107,115] It is a common prac-
tice in the isolation of LP that multiple separation tech-
niques, such as TLC and PTLC are used to obtain pure
compounds.[116]
High-performance chromatography (HPLC)
HPLC is a specific form of column chromatography used
in chemical and biochemical analysis. HPLC is a tech-
nique that can separate a mixture of surface active com-
pounds and is used to identify, quantify and purify
CRITICAL REVIEWS IN BIOTECHNOLOGY 401
individual components of the BS mixture. This technique
is gaining popularity among various chromatography
approaches and is usually the main choice for LP stud-
ies. The resolving power of HPLC is ideally suited to the
rapid processing of multi-component samples on both
analytical and preparative scales. HPLC is generally per-
formed using C8 or C18 reverse-phase columns with a
water/acetonitrile (ACN) gradient. Common detection
methods for the samples collected from HPLC columns
are UV spectroscopy, measurement of refractive index,
fluorescence, electrical conductivity and MS.
The use of HPLC has been reported in the character-
ization, quantification and purification of
LP.[12,117–119] For example, purification of LP by HPLC
was carried out by reversed phase (RP)-HPLC using a
semi-preparative C18 column and 0.1% trifluoroacetic
acid/methanol/H2O as a mobile phase. The elution was
monitored using UV detection at 214 and 280 nm. The
metabolites eluting as individual peaks were used for
further MS characterization.[120] Recently, the structure
of LP produced by Citrobacter and Enterobacter were
subjected to further characterization.[17] The LP sam-
ples were purified with a RP-HPLC C18 column and the
elution was also monitored by UV detection at 215 nm.
The solvent system used was (A) 0.1% aqueous TFA and
(B) ACN containing 0.1% TFA. The following gradient of
solvent B was used to run the column: 0–60% for
0–45 min, 60–80% for 45–55 min, and 80–100% for
55–60 min.
Liquid chromatography – mass spectroscopy
(LC-MS)
LC and MS, when used in tandem, provide a unique
capability for rapid, cost-effective and quantitative
measurements of organic molecules in an enormous
variety of applications. Coupling LC with MS provides
the advantages of characterizing retention time of a
given LP/BS along with its mass spectral signature,
allowing the analysis of complex mixtures. This is nor-
mally achieved by splitting the HPLC eluent to convey a
fraction of the eluent into the mass spectrometer.[1]
Direct analysis of LP/BS in fermentation broths using LC-
MS is possible, but it is usually necessary to perform ini-
tial steps of purification to remove the worst interfer-
ences and also to concentrate the sample if BS is only
present at very low concentrations. The most important
and widely used LC separation technique for quantita-
tive LC-MS is RP separation. This utilizes differences in
hydrophobicity (termed ‘‘nonspecific hydrophobic inter-
actions’’) to achieve partitioning between an apolar sta-
tionary phase and a polar mobile phase. Typically,
mobile phases are different blends of water with a
402 P. BINIARZ ET AL.

Table 4. HPLC solvent systems and MS approaches designed for the analysis of LP produced by microorganisms. Solvent mixtures
compositions are shown in volume ratios
MS method/
Biosurfactant Column Solvent system, flow rate RT from HPLC (min) ionization References
Surfactin Fengycin Analytical Luna 5 lm C18, (A) water, 1% formic acid; (B) ACN; C13 surfactin 18.89 [87]
A Fengycin B 150 mm  4.6 mm linear gradient A:B (50:50) for 3 min, C14 surfactin 20.06 ESI-MS
then A:B (0:100) over 18 min and C15 surfactin 20.06 [M þ H]þ
then 100% B over 5 min; flow rate of C14 fengycin A 3.80 [M  H]
0.8 mL/min C15 fengycin A 5.00 [M þ Na]þ
C16 fengycin A 6.50
C17 fengycin A 7.49
C14 fengycin B 4.94
C15 fengycin B 6.24
C16 fengycin B 7.41
C17 fengycin B 8.18
Lichenysin RRHD Zorbax Eclipse Plus (A) 2 mm ammonium acetate and ND ESI-MS [56]
1.8 lm C18 column, 0.2% heptafluorobutyric acid in water; [M þ H]þ
100 mm  2.1 mm (B) ACN and methanol (1:1); linear [M þ Na]þ
gradient from 90 to 93% B in 4 min,
8 min analysis time; flow rate
0.4 ml/min
Norsurfactin TOSOH TSKgel ODS- (A) distilled water with 0.1% formic ND ESI-MS [112]
Fengycin Iturin A 100 V 5 lm column, acid; (B) ACN containing 0.1% formic [M þ H]þ
Mixirin Pumilacidin 250 mm  4.6 mm acid; linear gradient from 3 to 97% B
Surfactin during 45 min; flow rate 0.5 mL/min

Iturin Surfactin Zorbax 300SB 5 mm ACN:water, both with 0.1% formic ND ESI-MS [121]
C18 column, acid as mobile phase solvents with ESI-MS/MS
150 mm  4.69 mm a 40 min linear gradient of 50–95% [M þ H]þ
ACN at 0.2 mL/min flow rate [M þ Na]þ
Bacillomycin F C18 reverse phase (RP) Linear gradient of deionized water ND ESI-MS [94]
column and ACN (0–100%) containing 0.05% ESI-MS/MS
trifluoroacetic acid [M þ H]þ
[M þ Na]þ
Viscosin group, Analytical Luna 5 mm C18, (A) 5 mmol/L ammonium acetate in Fraction A 6.5 ESI-MS [122]
cyclic LP 250 mm  4.60 mm water; (B) ACN, linear gradient from Fraction B 8.1/8.3 [M þ H]þ
a 25:75 ratio to a 0:100 ratio over a Fraction C 9.2 [M þ H]
time span of 15 min; flow rate of
1 mL/min.

miscible polar organic solvent, for example, ACN or
methanol. Table 4 shows several LC-MS systems
designed for the analysis of LP.
LC-MS techniques are also highly efficient in separat-
ing and purifying LP isoforms. Thus, an efficient high-re-
solution LC-MS method is a prerequisite for the
purification of microbial LP, which is required for subse-
quent commercialization of a particular LP isoforms as a
potential therapeutic agent.[123] LC-MS is best suited for
a discovery-based approach when working on unknown
LP, as many BS are readily amenable to LC-MS analysis.
Gas chromatography (GC)–MS
GC is widely used in applications involving the structural
analysis of BS. Typical applications pertain to the quanti-
tative and/or qualitative analysis of fatty acid structures.
When coupled to MS, it is possible to obtain additional
information about the molecular mass of each sepa-
rated compound and its elemental composition, func-
tional groups, and, in certain cases, molecular geometry
and spatial isomerism. The efficient GC-MS methods
generally require a relatively pure LP sample. The major-
ity of LP analyzed by GC-MS requires chemical derivati-
zation involving hydrolytic cleavage of the bond
between the peptide and lipid portions of LP to provide
volatility and thermal stability prior to analysis. The ana-
lysis of LP involves hydrolytic cleavage of the bond
between the peptide part and lipid portions of LP.
Subsequent derivatization of resulting fatty acid chains
to fatty acid methyl esters would facilitate GC or GC-MS
analysis.[124] GC-MS provides greater sensitivity than
LC-MS for free fatty acids as the high resolution of GC
permits separation of structurally similar fatty acids that
would be very difficult to separate by HPLC. GC is not
commonly used for the analysis of biomolecules since
samples (e.g. peptides) are thermally destroyed after
hydrolysis. Smaller molecules, such as amino acids, fatty
acids and certain carbohydrates, can be analyzed if they
are chemically modified to increase their volatility. While
GC is more sensitive, LC is more versatile as it is not
restricted to volatile and heat-stable samples; the LC
sample only has to dissolve completely in the mobile
phase.

GC-MS techniques have been used for resolving the
structure of LP produced by B. thuringiensis.[125] GC-MS
was utilized in the identification of fatty acid moieties in
purified LP where the LP was discovered to contain a
C17 fatty acid with one double bond between carbons
13 and 14.[125] In a study using GC-MS to analyze TLC-
purified LP, the GC analysis spectrum indicated that
fatty acids portions of the LP contained 3-hydroxy tride-
canoate (3-OH-C13), tetradecanoate (3-OH-C14), pentade-
canoate (3-OH-C15) and hexadecanoate (3-OH-C16).[126]
A subsequent study identified GC-MS peaks that were
characteristic of b-hydroxy fatty acid methyl deriva-
tives.[127] The purified active compound was hydro-
lyzed with 6 M HCl at 110  C for 2 h in a sealed vial. Free
fatty acids were converted to methyl esters and ana-
lyzed with a GC-MS. Peaks observed at m/z 74 and 103
(base peak) were characteristic for methyl derivatives of
b-hydroxy fatty acids.
Matrix-assisted laser desorption-ionization (MALDI)
time-of-flight (TOF) MS
MALDI MS is a soft ionization MS technique that allows
the identification of intact compounds. In MALDI, an
analyte is first co-crystallized with a large molar excess
of a matrix compound, typically a UV-absorbing weak
organic acid. The mixture is subsequently exposed to
laser radiation, resulting in the vaporization of the
matrix that carries the analyte with it. The matrix com-
pound plays a key role by strongly absorbing the laser
light energy, indirectly causing the analyte to vapor-
ize.[128] The matrix compound also serves as a proton
donor and acceptor and forces the analyte to ionize in
both the positive and negative ionization modes,
respectively.[129] MALDI can be used to determine the
full molecular mass of purified LP/BS. Easy sample prep-
aration and recent developments in small bench top
MALDI-TOF MS instruments have made this an attractive
technique for LP analysis. A recent report utilized
MALDI-TOF MS to identify surfactin LP.[17] In MS/MS
sequencing, the lactone ring present in LP was cleaved
by incubating each peptide with 10% NaOH in metha-
nol at room temperature for 16 h. The mass spectra of
LP showed a series of mass number of m/z 1043
and assigned it to the LP group surfactin with m/z 637
and 985.
NMR analysis of LP
NMR spectroscopy is an experimental method that can
provide structural information of molecules in solution
with a high resolution. NMR spectroscopy is a high-
throughput technique. Structure determination by NMR
CRITICAL REVIEWS IN BIOTECHNOLOGY 403
may be divided into the following steps: (1) Establishing
suitable conditions for recording spectra; (2) Measuring
a series of 1D (1H and 13C) or 2D (e.g. COZY, TOCSY and
ROSY) NMR spectra; (3) Integrating cross peaks and
transformation into upper-distance bounds (calibration);
(4) Assessing the quality of the molecular structure.
Samples are mixed with a reference compound solution
(e.g. tetramethylsilane dissolved in DMSO-d6 for 1H
NMR), added to an NMR probe (generally less than
2 ml), inserted into the instrument, generating the NMR
spectrum for analysis.
NMR can be used to confirm LP structural identifica-
tion of both the peptide and fatty acid portion while
simultaneously providing data on the position of link-
ages between the peptide and fatty acid chain. For LP
NMR experiments, purified LP is dissolved in deuterated
chloroform, and a series of 1D and 2D NMR experiments
are carried out.[30] Results from NMR spectroscopy are
drawn from the NMR spectrum which depends on the
effect of shielding by electrons orbiting the nucleus.
Chemical shifts in the spectrum represent alterations or
changes in the molecular structure. The chemical shift
for 1H NMR (Figure 2A) is determined as the difference
(in ppm) between the resonance frequency of the
observed proton and that of a reference proton present
in a reference compound set at 0 ppm. All 1D and 2D 1H
NMR spectra of LP produced by B. licheniformis were
recorded at 299 K locked to the deuterium resonance of
the solvent, DMSO-d6 (Figure 2B).[110] The NMR data
indicated that the peptide moiety contained seven
amino acids per molecule. Complete amino acid spin
systems and amino acid sequence were identified from
a 2D 1H phase. NMR spectra indicated the presence of a
long chain fatty acid, which contained a b-hydroxyl
group.
The power of sophisticated techniques, such as
NMR spectroscopy for structural elucidation is
extremely appealing. It is tempting to believe that
structures determined by NMR application are
unequivocal. A common mistake is to use a 13C NMR
carbon count to determine the molecular formula for a
LP. Although the elemental composition can be pre-
sumed by this procedure, it has not been conclusively
established.[131]
FT-IR
FT-IR spectroscopy is a rapid and inexpensive method to
characterize the chemical structure of BS and identify
the functional groups present in LP. FT-IR is a physico-
chemical method based on measuring the vibrations of
a molecule excited by IR radiation at a specific wave-
length range. This technique determines the functional
404 P. BINIARZ ET AL.
Figure 2. (A) 1H chemical shift positions of chemical groups in biomolecules [130] and (B) 2D 1H-1H TOCSY spectrum of a
Pseudomonas LP [122]
Figure 3. FTIR spectrum of a LP/BS produced by Candida tropicalis MTCC23 [133]
groups of gases, liquids, and solids and gives a structural
elucidation of a compound of interest. The presence or
absence of functional groups, their protonation states,
or any changes due to new interactions can be moni-
tored by analyzing the position and intensity of different
IR absorption bands. Despite some differences in FT-IR
spectra among LP or experimental conditions, the IR
spectrophotometer is used in the range between
approximately 4000 and 400 cm1.[132]
The interpretation of spectra and peak assignments
are key steps in the FT-IR analysis of LP. The wave num-
ber positions of absorbance peaks, peak intensities and
peak widths are useful for functional group and sample
identification. There are several interesting peaks that
appear on an IR spectrum of a LP (Figure 3 and Table
S3), which are considered to be characteristic of
LP.[134,135]
As with any analytical techniques, FT-IR approaches
have certain advantages and disadvantages. FT-IR is
advantageous in that an IR spectrophotometer and the
necessary software are readily available and can be
used for routine analysis. In addition, FT-IR methods are
relatively fast, simple to use, and cheap and IR spectros-
copy is nondestructive, i.e. the structure of LP remains
unchanged during analysis. However, environmental
conditions around the FT-IR instrument can cause varia-
tions in the spectra. Hence, background scans and
repeated scans of the same sample are required. In add-
ition, mixtures of BS may exhibit overlapping spectra,
leading to a misinterpretation of results; therefore, a
proper purification step is required prior to performing
FT-IR.
FT-IR has been utilized in LP studies to identify the
type of LP. For example, FT-IR analysis of purified LP

produced by B. circulans and standard surfactin identi-
fied the purified LP as surfactin.[136] The samples were
dispersed in spectral grade KBr and measurements were
carried out in the transmittance mode in the
400–4000 cm1 range. The FT-IR spectrum of the puri-
fied compound showed transmittance at 1260 and
1900 cm1 that may be present due to the stretching
vibrations of C–O and C¼O. A single peak at 1590 cm1
corresponded to the deformation of N–H bonds, and a
strong NH bond was observed at 3420 cm1. FT-IR spec-
tra of the purified BS also has closely resembled the IR
absorption pattern displayed by other LP like surfac-
tin,[90] fengycin,[19] iturin [106] and bacillomycin.[137]
The presence of lichenysin A in B. licheniformis BAS50
was confirmed using FT-IR.[110] Huang et al. [34] con-
ducted a quantitative analysis of LP using FT–IR spectra,
which indicated the presence of the ester group.
Conclusions and perspectives
Intensive development in biotechnology, genetic engin-
eering and an increase in human responsibility for envir-
onmental protection contribute to the search for new
microorganisms producing biomolecules that have
desirable characteristics and would be useful in a multi-
tude of fields. BS have the potential to be such an all-
purpose molecules in the future.
Over the years, BS and LP in particular have become
broadly pertinent in various industries and, therefore,
pose a serious alternative to synthetic surfactants.
However, it will be imperative to evaluate the major
applications of LP and to determine whether the large-
scale production of quality LP is feasible. LP are promis-
ing natural molecules that may one day have wide-
spread use, for example, as therapeutic agents in
medicine or in bioremediation to assist waste disposal.
Crude LP can be immediately used in bioremediation
applications, and partially purified fractions (about
60–80% pure) can suit applications in the laundry and
food industry. However, if LP are to be considered for
pharmaceutical and human healthcare applications, the
requirement for ultra-pure product is indispensable.
With exciting new developments in LP research, the
focus on chromatographic (e.g. HPLC and GC) and spec-
troscopic techniques (e.g. MS, NMR and FT-IR) combined
with technologies of large-scale fermentation and gen-
etic and metabolic engineering suggest that LP will be
commercially successful compounds in the future.
The promising future of LP/BS in a variety of fields
also depends greatly on discovering BS with desirable
activities. Technology is rapidly advancing, providing
suitable techniques to facilitate LP/BS studies. However,
none of techniques that are currently exploited for
CRITICAL REVIEWS IN BIOTECHNOLOGY 405
screening and initial steps of BS production, purification
and characterization is the ideal one.
In this review, we have presented a thorough investi-
gation of various means of screening, production, isola-
tion, purification and structural characterization of LP.
Rapid advances in the last few years have helped us to
understand the process of LP production by microor-
ganisms. This knowledge will serve to increase the rate
at which novel LP/BS can be identified and character-
ized to identify the molecules that have desirable char-
acteristics and would be useful in a multitude of fields.
There are still drawbacks in the available techniques for
rapid LP study, therefore, this area should be pursued.
Acknowledgements
The authors would like to thank anonymous reviewers for
their comments and help with improving this manuscript.
This work was supported by Wroclaw Center of
Biotechnology, program: The Leading National Research
Center (KNOW) for years 2014–2018.
Disclosure statement
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of the paper.
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