Rhamnolipids—brief outline
Biosurfactants, widely known as surface-active agents of biological origin, have carved
a niche for themselves in the market due to their unique environment-friendly properties.
They have come a long way since first biosurfactant “surfactin” was purified and characterized
by Arima et al. (1968). Biosurfactants have been researched thoroughly and satisfactorily
since then by many research groups across the world yet there are aspects that elude
our understanding. There are five major categories of biosurfactants viz. glycolipids,
phospholipids and fatty acids, lipopeptides and lipoproteins, polymeric biosurfactants
and particulate biosurfactants that have found applications in agricultural, pharmaceutical,
food, cosmetics, and detergent industries. Data reveals there are more than 250 patents
obtained on these wonder biodegradable molecules so far (Shete et al., 2006; Rahman
and Gakpe, 2008). It has also been observed that microbial biosurfactants are advantageous
over plant-based surfactants because of the scale-up capacity, rapid production, and
multi-functional properties. Several plant-based biosurfactants for example saponins,
lecithins, and soy proteins have excellent emulsification properties but are expensive
to produce at industrial scale and have other debatable issues such as solubility
and hydrophobicity (Xu et al., 2011).
Among the various categories of biosurfactants the glycolipid biosurfactants “rhamnolipids”
stand apart. Rhamnolipid, primarily a crystalline acid, is composed of β-hydroxy fatty
acid connected by the carboxyl end to a rhamnose sugar molecule. Rhamnolipids are
predominantly produced by Pseudomonas aeruginosa and classified as: mono and di-rhamnolipids.
Other Pseudomonas species that have been reported to produce rhamnolipids are P. chlororaphis,
P. plantarii, P. putida, and P. fluorescens. Some bacteria are known to produce only
mono-rhamnolipids while some produce both. The ratio of mono and di-rhamnolipid can
also be controlled in the production method. There are enzymes available that can
convert mono-rhamnolipids into di-rhamnolipids. In 1984, the first patent for the
production of rhamnolipids was filed by Kaeppeli and Guerra-Santos (US 4628030) and
obtained in 1986 for their work on Pseudomonas aeruginosa DSM 2659 (Kaeppeli and Guerra-Santos,
1986). Subsequently, Wagner et al. filed a patent (US 4814272) in 1985 for the biotechnical
production of rhamnolipids from Pseudomonas sp. DSM 2874 and obtained the same in
1989 (Wagner et al., 1989). In the past close to three decades, there has been a great
body of research work carried on rhamnolipids revealing many of their astonishing
applications and making them reach the pinnacle of popularity among all the categories
of biosurfactants in the global market. The reason behind the current global interest
in rhamnolipid production owes to their broad range of applications in various industries
along with many spectacular “eco-friendly” properties.
The current critique articulates to present opinion on rhamnolipid research and is
an attempt to retrospect what brings rhamnolipids in the forefront. This article is
a bird's-eye view on a timeline of rhamnolipids story so far and also a critical analysis
on why despite so many patents and research work rhamnolipids still do not rule the
global biosurfactant market.
Inimitable applications of rhamnolipids
Over the years rhamnolipids are becoming broadly pertinent in various industries and
are posing a serious threat to the synthetic surfactants. Before venturing into the
current production economics of rhamnolipids it is imperative to evaluate the major
applications of rhamnolipids that make them noticeable among other biosurfactants.
A list of five major applications of rhamnolipids that cater to the wide range of
industrial demands includes:
Bioremediation and enhanced oil recovery (EOR): Rhamnolipids show excellent emulsification
properties, efficiently remove crude oil from contaminated soil and facilitate bioremediation
of oil spills (Rahman et al., 2003; Costa et al., 2010).
Pharmaceuticals and therapeutics: Rhamnolipids show low toxicity, surface active properties
and antimicrobial activities against several microbes (Bacillus cereus, Micrococcus
luteus, Staphylococcus aureus, Listeria monocytogenes) thereby showing promising applications
in pharmaceuticals and therapeutics (Magalhaes and Nitschke, 2013).
Cosmetics: Rhamnolipid as an active ingredient is found to be effective for several
skin treatments i.e., wound healing with reduced fibrosis, cure of burn shock, treatment
of wrinkles hence are in demand in the health and beauty industry (Piljac and Piljac,
2007).
Detergents and cleaners: Rhamnolipids are natural emulsifiers and surface active agents
leading to their wide spread usage in detergent compositions, laundry products, shampoos
and soaps (Parry et al., 2013).
Agriculture: Rhamnolipids are already used for soil remediation for improving soil
quality and are now further getting explored for plant pathogen elimination, for aiding
the absorption of fertilizers and nutrients through roots and as biopesticides (Sachdev
and Cameotra, 2013).
Biosurfactant producing companies—with focus on rhamnolipids
Rhamnolipids are highly applicable in various activities with some researchers advancing
the technology from laboratory to higher scale. However, there still are very limited
companies in the field which are producing biosurfactants at a marketable scale. We
tried to compile a list of biosurfactant producing companies around the globe (Table
1). The compilation evidently defines which biosurfactants are mostly researched and
produced at higher scale.
Table 1
Biosurfactant producing companies around the globe.
S. No.
Company
Location(s)
Product(s)
Focus on
1
TeeGene Biotech
UK
Rhamnolipids and Lipopeptides
Pharmaceuticals, cosmetics, antimicrobials and anti-cancer ingredients
2
AGAE Technologies LLC
USA
Rhamnolipids (R95, an HPLC/MS grade rhamnolipid)
Pharmaceutical, cosmeceutical, cosmetics, personal care, bioremediation (in situ &
ex situ), Enhanced oil recovery (EOR)
3
Jeneil Biosurfactant Co. LLC
USA
Rhamnolipids (ZONIX, a bio-fungicide and RECO, a rhamnolipid used in cleaning and
recovering oil from storage tanks)
Cleaning products, EOR
4
Paradigm Biomedical Inc.
USA
Rhamnolipids
Pharmaceutical applications
5
Rhamnolipid Companies, Inc.
USA
Rhamnolipids
Agriculture, cosmetics, EOR, bioremediation, food products, pharmaceutical
6
Fraunhofer IGB
Germany
Glycolipids, Cellobiose lipids, MELs
Cleansing products, shower gels, shampoos, washing-up liquids, pharmaceutical (bioactive
properties)
7
Cognis Care Chemicals
China, Germany, USA
Alkyl polyglucoside APG®, Plantacare 1200 GLY (green surfactant for use in oral-dental
formulations), Rheocare TTA (for cleansing formulations)
Used in formulations for household cleaners, bath/shower gels, dish washing, laundry
detergents and in agrochemicals
8
Saraya Co. Ltd.
Japan
Sophorolipids (Sophoron, a low-foam dishwasher detergent)
Cleaning products, hygiene products
9
Ecover Belgium
Belgium
Sophorolipids
Cleaning products, cosmetics, bioremediation, pest control, pharmaceuticals
10
Groupe Soliance
France
Sophorolipids
Cosmetics
11
MG Intobio Co. Ltd.
South Korea
Sophorolipids (Sopholine—functional soap with Sophorolipids secreted by yeasts)
Beauty and personal care, bath supplies e.g., soaps with new functions
12
Synthezyme LLC
USA
Sophorolipids
Cleaning products, cosmetics, food products, fungicides, crude oil emulsification
13
Allied Carbon Solutions (ACS) Ltd
Japan
Sophorolipids (ACS-Sophor—first bio-based surfactant from Indian mahua oil)
Agricultural products, ecological research
14
Henkel
Germany
Sophorolipids, Rhamnolipids, Mammoslyerthritol lipids
Glass cleaning products, laundry, beauty products
15
Lion Corporation
Japan
Methyl ester sulfonates (MES)
Detergents formulations, cleaning products
16
Lipo Chemicals
USA
Lipomulse Luxe (high-temperature resistance emulsifier)
Skin care, sun-lotions hair care formulations, thickening polymers, rheological modifiers,
natural gums
17
Kaneka Co.
Japan
Sophorose lipids
Cosmetics and toiletry products
Availability of feedstock and its impact on biosurfactants
Biodiesel is produced by the trans-esterification of vegetable oils and fats with
methanol in the presence of a catalyst. Glycerol is received as a by-product from
this reaction. The production of 1 ton biodiesel generates about 100 kg of glycerol.
Hence, the European biodiesel industry might release about 600 Kiloton glycerol per
year with an increasing tendency in Europe and worldwide. Oversupply of glycerol,
essentially due to increasing biodiesel production, leads to decreasing prices and
weak markets.
The price of pure glycerol varied from $0.50 to $1.50/lb and crude glycerol from $0.04/kg
to $0.33/kg over the past few years. The price of glycerol in the market will continue
to drop in such an over saturated market. Currently, the main supply of glycerol coming
into the market is from the rapidly growing biodiesel industry. Estimated production
of glycerol would reach 5.8 billion pounds in 2020. This is due to demand of biodiesel
that is projected at 8 billion gallons in 2020 (Ayoub and Abdullah, 2012). Hence new
outlets for glycerol are urgently needed, particularly in the case of crude glycerol
released by the biodiesel processes. As glycerol is a nontoxic, edible, biodegradable
compound, it will provide important environmental benefits to the new platform products.
In case of biosurfactant production, dramatically rising in biodegradable, non-toxic
and eco-friendly alternative for chemical surfactants and the re-discovered opportunity
of biosurfactants that gave rise to invention and investment ahead of the typical
rigors of techno-economic modeling for the use of glycerol as a feed stock, leading
typically to unmet expectations. Bacteria produce biosurfactants if grown on carbon
sources such as glucose, glycerol, and various vegetable oils. Our research on biosurfactant
production by bacteria indicates that glycerol can be used efficiently for biosurfactant
production (Rahman et al., 2002).
The considerable interest in biosurfactants in the recent years is also due to their
low toxicity, biodegradable nature and specificity, which would be very suitable to
meet the European Surfactant Directive. Regulation EC No.: 648/2004 requires clear
and precise description of the biodegradability of the surfactant and test methods
to give assurance of its aerobic biodegradability. This regulation establishes rules
designed to achieve the free movement of detergents and surfactants for detergents
in the internal market while, at the same time, ensuring a high degree of protection
of the environment and human health.
Surfactants constitute an important class of industrial chemicals and are widely used
in almost every sector of modern industry. Most of the commercially available surfactants
are chemical surfactants mainly, petroleum-derived. However, rapid advances in biotechnology
and increased environmental awareness among consumers combined with expected new environmental
legislation has provided further impetus for serious consideration of biological surfactants
as possible alternatives to existing products.
Biosurfactant's economic feasibility—what it takes to become a market leader?
As described in the previous section, there is enormous awareness among the consumers
these days with regard to sustainability and global warming. The demand for bio-based
technologies is ever increasing and “green solutions” are sought for every process.
Rhamnolipids have promising properties and fulfill the eco-friendly criteria, one
of the main drivers, but are still struggling to become market leaders. The economics
of production is a major bottleneck in the outburst of commercialization of rhamnolipids
and other biosurfactants (Table 2). There is still no downstream technology economical
and convincing enough to recover and purify rhamnolipids at industrial scale. In case
of biosurfactant production the downstream processing accounts for 70–80% of the entire
production costs.
Table 2
Cost of biosurfactant per liter of solution (diluted and the CMC based cost calculation
carried out by Connolly et al., 2010).
Biosurfactant
Origin
Supplier
ST mN/m
CMC (%)
Cost (£/L)
BioFuture
Bacterial rhamnolipid
BioFuture Ltd. Dublin
28
0.08
0.02
Citrasolv
Orange peel
Cleveland Biotech Ltd., Teesside
30
0.9
0.01
EC601
Bacterial rhamnolipid
Ecochem Ltd., Canada
29
0.2
0.23
EC1800
Bacterial consortium
Ecochem Ltd., Canada
28
0.04
0.01
Petrosolv
Bacterial unknown
Enzyme Technologies Inc., USA
34
0.2
0.01
Saponin
Plant bark
Sigma UK
45
0.1
0.50
The table also gives the origin of biosurfactant along with surface tension (ST) and
critical micelle concentration (CMC) values.
It is a no-brainer that in order to gain higher profit at commercial scale it requires
access to very cheap feedstock. There are some other key parameters that need thorough
consideration in order to make any product economically feasible. Technological fit
and process optimization are among the main drivers. Fermentation time is another
key to success. Fermentation performance and scale impact process economy as it is
directly related to the yield, titer, and productivity. High cost of production especially
because of the expensive substrates and down-stream processes makes it difficult to
bring down the price of these environment friendly biomolecules. In order to compete
with the synthetic detergents or surfactants the cost of production must be brought
down to £1.70 per liter which is in itself a challenging task. As there are many barriers
in the commercialization of biosurfactants, there seems no dearth of opportunities
in this field. Cost comparison of various technologies viz. enzymatic, continuous,
shake flask, batch, and fed-batch used for biosurfactant production pinpoint the requirement
of innovative methods wherein rhamnolipids can be produced in static conditions to
reduce the fermentation cost. The operating costs can be brought down by robust wild-type
strains or recombinant mutant strains. Testing the possibility of co-products and/or
enzymes is another attractive solution to surge the net profit—for example: esterases
released during the production of lipopeptides by Bacillus strain and its recombinants
(Sekhon et al., 2011, 2012). Co-products and by-products are value drivers and increase
the economic viability of any business. The search of cheap and easily accessible
raw material or substrate for biosurfactants production has been going on for years.
The utilization of by-products, even if from a different process could be another
smart solution—for example: glycerol, which is a by-product of biodiesel production,
is available in surplus amount in the global market (Albarelli et al., 2011) which
might be a cheap alternative for biosurfactant production.
Rhamnolipids are well-characterized and scientifically proven biosurfactants which
are slowly and steadily becoming highly sought after biomolecules. Among other biosurfactants
rhamnolipids have the highest number of patents (Table 3) and research publications.
However, cost-competitiveness is one of the major factors that is holding rhamnolipids
back from becoming the champions of their field. Research needs to be focused on suitable
vigorous production strains, cheap substrates and nominal bioreactor technology. The
current market price of rhamnolipid (R-95, 95%) is $227/10 mg (Sigma-aldrich) and
$200/10 mg (AGAE technologies, USA) calling for strenuous research. Rhamnolipids have
favorable applications in various sectors and if made economically sustainable nothing
can stop these biomolecules to rule the surface-active compounds market.
Table 3
A timeline and the major patents and grants obtained on rhamnolipids so far.
S. No.
Patent or Application No.
Filed
Issued
Title
Inventors
1
4628030
Aug 1984
Dec 1986
Process for the production of rhamnolipids
Kaeppeli and Guerra-Santos
2
4814272
Feb 1985
March 1989
Process for the biotechnical production of rhamnolipids including rhamnolipids with
only one. Beta.-hydroxydecanoic acid residue in the molecule
Wagner et al.
3
4933281
March 1987
June 1990
Method for producing rhamnose
Daniels et al.
4
4902512
Jan 1988
Feb 1990
Rhamnolipid liposomes
Ishigami et al.
5
5417879
Sep 1993
May 1995
Synergistic dual-surfactant detergent composition containing sophoroselipid
Hall et al.
6
5455232
April 1994
Oct 1995
Pharmaceutical preparation based on rhamnolipid
Piljac and Piljac
7
5550227
May 1994
Aug 1996
Method for the preparation of rhamnose monohydrate from rhamnolipids
Mixich et al.
8
5466675
July 1994
Nov 1995
Immunological activity of rhamnolipids
Piljac and Piljac
9
5520839
Jan 1995
May 1996
Laundry detergent composition containing synergistic combination of sophorose lipid
and non-ionic surfactant
Hall et al.
10
5501966
Jan 1995
March 1996
Pseudomonas aeruginosa and its use in a process for the biotechnological preparation
of L-rhamnose
Giani et al.
11
5658793
June 1995
Aug 1997
Pseudomonas aeruginosa and its use in a process for the biotechnological preparation
of L-rhamnose
Giani et al.
12
5514661
Aug 1995
May 1996
Immunological activity of rhamnolipids
Piljac and Piljac
13
5767090
Jan 1996
June 1998
Microbially produced rhamnolipids (biosurfactants) for the control of plant pathogenic
zoosporic fungi
Stanghellini et al.
14
7129218
Aug 2000
Oct 2006
Use of rhamnolipids in wound healing, treatment and prevention of gum disease and
periodontal regeneration
Stipcevic et al.
15
7262171
Aug 2000
Aug 2007
Use of rhamnolipids in wound healing, treating burn shock, atherosclerosis, organ
transplants, depression, schizophrenia and cosmetics
Piljac and Piljac
16
20040224905
May 2002
Nov 2004
Use of rhamnolipids in wound healing, treatment and prevention of gum disease and
periodontal regeneration
Stipcevic et al.
17
20060233935
Nov 2003
Oct 2006
Rhamnolipids in bakery products
Haesendonck and Vanzeveren
18
7202063
Aug 2005
April 2007
Processes for the production of rhamnolipids
Gunther et al.
19
20070191292
Feb 2006
Aug 2007
Antimycotic rhamnolipid compositions and related methods of use
Gandhi et al.
20
20070155678
Feb 2007
July 2007
Use of rhamnolipids in wound healing, treating burn shock, atherosclerosis, organ
transplants, depression, schizophrenia and cosmetics
Piljac and Piljac
21
20070207930
Feb 2007
Sep 2007
Rhamnolipid compositions and related methods of use
Gandhi et al.
22
7968499
Feb 2007
June 2011
Rhamnolipid compositions and related methods of use
Gandhi and Skebba
23
20080213194
July 2007
Sep 2008
Rhamnolipid-based formulations
Keith DeSanto
24
7985722
July 2007
July 2011
Rhamnolipid-based formulations
Keith DeSanto
25
20100249058
Oct 2007
Sep 2010
Feed additive and feed
Ito et al.
26
20090126948
Nov 2007
May 2009
Use of rhamnolipid based formulations for fire suppression and chemical and biological
hazards
Keith DeSanto
27
20080261891
Feb 2008
Oct 2008
Compositions and methods for using syringopeptin 25A and rhamnolipids
Bart C. Weimer
28
20090220603
May 2009
Sep 2009
Use of rhamnolipids in wound healing, treating burn shock, atherosclerosis, organ
transplants, depression, schizophrenia and cosmetics
Piljac and Piljac
29
20110123623
Nov 2010
May 2011
Rhamnolipid mechanism
Keith DeSanto
30
20120322751
Feb 2011
Dec 2012
Use of rhamnolipids as a drug of choice in the case of nuclear disasters in the treatment
of the combination radiation injuries and illnesses in humans and animals
Goran Piljac
31
20110257115
June 2011
Oct 2011
Method for treating rhinitis and sinusitis by rhamnolipids
Anton Leighton
32
20110306569
June 2011
Dec 2011
Rhamnolipid biosurfactant from Pseudomonas aeruginosa strain NY3 and methods of use
Yin et al.
33
8592381
June 2011
Nov 2013
Method for treating rhinitis and sinusitis by rhamnolipids
Anton Leighton
34
20110270207
July 2011
Nov 2011
Rhamnolipid based formulations
Keith DeSanto
35
8183198
July 2011
May 2012
Rhamnolipid-based formulations
Keith DeSanto
36
20130130319
July 2011
May 2013
Cells and methods for producing rhamnolipids
Schaffer et al.
37
20120255918
April 2012
Oct 2012
Use of rhamnolipids in the water treatment industry
DeSanto and Keer
38
20130296461
May 2013
Nov 2013
Aqueous coatings and paints incorporating one or more antimicrobial biosurfactants
and methods for using same
Lakshmi Sadasivan
39
20130310330
July 2013
Nov 2013
Method for treating obesity
Anton Leighton
40
8765694
July 2013
July 2014
Method for treating obesity
Anton Leighton
41
20140080771
Nov 2013
March 2014
Method for treating rhinitis and sinusitis by rhamnolipids
Anton Leighton
42
20140148588
Nov 2013
May 2014
Process for the isolation of rhamnolipids
Schilling et al.
Concluding remarks
As the Health and Safety in the bioprocessing become paramount for large scale production
there are significant interests in the search for novel non-pathogenic rhamnolipid
producers. The numbers of cultured organisms from the environmental samples are only
a tiny fraction (0.001%) of the actual microbial diversity. There are significant
number of microbial isolates that needs to be explored and exploited for rhamnolipid
and other bioproduct manufacturing. Biosurfactant producing probiotic organisms will
play a key role in the future of biosurfactant market. Edible emulsifiers from these
processes would be applicable to many applications including food, cosmetic, environmental
clean-up, biomedical and natural therapy. Rhamnolipid could be a potential alternative
for the synthetic surfactant molecules and an important platform chemical cluster
with the market value of $2.8 billion in 2023 (Grand View Research Inc., 2014). There
is a significant need for the discovery of novel non-pathogenic rhamnolipid producers
with enhanced production capacity and efforts to scale up through bioprocess engineering
are important to meet the future predictions of biosurfactants market.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.