Process Biochemistry 100 (2021) 124–139

Contents lists available at ScienceDirect

Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Review

Microbial-derived glycolipids in the sustainable formulation of biomedical

and personal care products: A consideration of the process economics
towards commercialization
Chukwudi O. Onwosi *, Godwin O. Aliyu , Chisom J. Onu , Kenechi O. Chukwu ,
Johnson K. Ndukwe , Victor C. Igbokwe
Department of Microbiology, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria

A R T I C L E I N F O A B S T R A C T

Keywords: Biosurfactants – tensioactive biometabolites – are currently preferred over their synthetic counterparts due to
Biosurfactants some unique properties such as sustainable production, eco-compatibility, and low cytotoxicity, among others.
Microbial-derived glycolipids Among different biosurfactant groups currently known, the microbial-derived glycolipids (MGLs) are of growing
Process economics
interests in the formulation of biomedical and cosmetic products due to their structural diversities, antimicrobial
Tensioactivity
Cosmetics
properties, stability under harsh environmental conditions, and functional versatility. While the usefulness of the
Biomedicine MGLs in biomedical and cosmetics fields cannot be overemphasized, their commercial competitiveness has been
restrained due to a myriad of bottlenecks which include poor product yields and high cost of production asso­
ciated with downstream processing. Herein, we reviewed the importance of process economics in MGLs pro­
duction to project strategies needed for product competitiveness in terms of yields and cost which will favour
their global market. The significance of techno-economic assessment in translating laboratory-based data to
commercial-scale MGL production was underlined. The strategies adopted by commercial MGL-producing firms
to enhance the market value of their products were also presented. Finally, the future of MGLs in the surfactant
market is highlighted.

1. Introduction make them indispensable in different biotechnological applications [2,

Surfactants are compounds that lower the surface tension (or inter­
facial tension) between two liquids, gas and liquid, or liquid and solid.
The majority of surfactants that find commercial applications in
biomedical and personal care products are from the synthetic origin
(mainly from petroleum derivatives). However, they do not support the
goals of green chemistry and sustainable production due to the eco-
hazardous impacts (e.g., skin irritation or damage) associated with
their long term use [1]. Hence there is a need for more viable and
eco-friendly alternatives such as the biosurfactants. Biosurfactants,
secreted by diverse microbes (fungi, bacteria, yeasts), are an interesting
group of secondary metabolites exhibiting tensioactive features which
3]. Among the biosurfactants, microbial-derived glycolipids (MGLs) are
the most popular in the global surfactant market. The MGLs family
comprises the rhamnolipid (RL), mannosylerythritol lipid (MEL), soph­
orolipid (SL), trehalolipid (TL), cellobiose lipids (CL) and xylolipids (XL)
[2,4]. In the past decade, MGLs are among the most sought-after bio­
metabolites towards the development of biomedical and personal care
(cosmetic) products [5]. This is due to their unique properties such as
low toxicity, ecological compatibility and biodegradability [2,6,7].
Other reasons include their excellent physicochemical attributes (e.g.,
tensioactivity, environmental stability), biochemical actions (e.g.,
cell-differentiation and antitumor activities), structural versatility and
sustainable production [2,4,7–10]. MGLs also exhibit significant

Abbreviations: AIST, National Institute of Advanced Industrial Science and Technology; Cc, characteristic curvature value; CL, cellobiose lipids; CMC, critical
micelle concentration; DC-Chol, 3 [N- (N′ , N′ –dimethylaminoethane)-carbamoyl] cholesterol; DOPE, L-dioleoylphosphatidylethanolamine; HLB, hydrophilic-lipo­
philic balance; HTS, high throughput screening; LRAC, long run average cost; MEL, mannosylerythritol lipid; MGL, microbial-derived glycolipids; OH-Chol, cho­
lesteryl-3β-carboxyamidoethylene-N-hydroxyethylamine; PodA, pyocyanin demethylase; R& D, research and development; RL, rhamnolipid; Sit-G, β-sitosterol β-D-
glucoside; SL, sophorolipid; TEA, techno-economic assessment; TL, trehalolipid; XL, xylolipids.
* Corresponding author.
E-mail address: chukwudi.onwosi@unn.edu.ng (C.O. Onwosi).

https://doi.org/10.1016/j.procbio.2020.10.001
Received 24 June 2020; Received in revised form 20 September 2020; Accepted 2 October 2020
Available online 7 October 2020
1359-5113/© 2020 Elsevier Ltd. All rights reserved.
C.O. Onwosi et al.
resilience to extreme conditions of salinity, temperature and pH [4,11,
12]. These interesting attributes extend to antibacterial, antiviral and
antifungal properties [4], which are of immense therapeutic and
biomedical importance [4,11,13].
While MGLs are promising in health care and cosmetics, some
technical hitches such as low productivity, highly heterogeneous prod­
ucts and strenuous downstream protocols have made their current prices
inevitably higher than the synthetic counterparts [8]. Thus, the
commercialization of MGL production has been unrealistic, despite the
enormous potential applications and properties [6,14,15]. The high
costs encountered during the production could be attributed to the
inherent costs of nutritive feedstocks and expensive downstream proc­
essing/purification [16,17]. For instance, some MGLs are produced by
pathogenic organisms (e.g., Pseudomonas aeruginosa which produces
RLs) which require extra purification steps due to biosafety concerns;
thus, leading to high cost of production. In other instances, the
complexity of regulation mechanisms (e.g., quorum sensing), low pro­
ductivity and difficulties in separation of the mixture of congeners,
hinder the scale-up of the production process [14,18,19]. Unfortunately,
these scenarios have made chemical surfactants outcompete MGLs in the
global surfactants market. To overcome these challenges and to meet the
demand in the biomedical and personal care products – which require
high purity and specific products – the MGL production process should
be more flexible to ensure cost-effectiveness. These adaptable bio­
processes include efficient and affordable integrated downstream pro­
tocols for enhanced volumetric productivity, low-cost feedstocks,
development of tailor-made products, etc. [20–22].
Although vast reports exist in the literature on the application of
MGLs in the formulation of biomedical and personal care products, none
have been dedicated to process economics necessary for commerciali­
zation. Therefore, the current review was undertaken to address this
knowledge gap. First off, we presented some of the interesting physi­
cochemical properties of the MGLs that make them relevant in the
biomedical and personal care applications. Emerging areas of applica­
tion of MGLs in biomedical and personal care products also formed part
of the current report. The scientific research and development (R & D)
that have been currently put in place to alleviate these concerns raised
on MGLs are also presented. We took a further step to summarize the
techno-economic assessments that are gainful in the scaling up of the
production of MGLs to industrial scale. The future of MGLs in the
development of health and personal care products is also presented.
2. Physicochemical properties of MGLs
Given the current state-of-the-art facilities, the structural configu­
rations of MGLs have been elucidated. This past decade has witnessed
the development of some robust techniques (strategies) for structural
elucidation of pre-existing and novel MGLs. These techniques comprise:
one- and two-dimensional nuclear magnetic resonance (1D and 2D
NMR) spectrometry, gas chromatography-mass spectrometry (GC–MS),
Fourier transform infrared spectroscopy (FTIR), matrix-assisted laser
desorption/ionization-time-of-flight mass spectroscopy (MALDI-TOF),
high performance-liquid chromatography (HPLC), thin layer chroma­
tography (TLC), liquid chromatography-mass spectrometry (LC–MS),
High-resolution electrospray ionization time-of-flight mass spectrom­
etry (HR-ESI-TOF-MS) [15,23–25].
The importance of MGLs in the sustainable formulation of bio­
technologically relevant products is due to their unique tensioactive
properties particularly the surface tension (determinant of effectiveness)
and critical micelle concentration (a strong indicator of efficiency).
From a practical standpoint, it is vital to underline that the efficiency
and effectiveness of MGLs are at variance. While effectiveness is deter­
mined by the lowest value that can be attained during surface tension
(ST) reduction, efficiency shows the concentration of surfactant that
brings about significant lowering of the ST of liquid (e.g., water) [26,
27]. Compared to the synthetic surfactants, the critical micelle
125
Process Biochemistry 100 (2021) 124–139
concentration (CMC) of MGLs is several times lower; which means that
less concentration of the latter will be required for effective ST reduction
[28].
Hydrophilic-lipophilic balance (HLB) is also a helpful physico­
chemical parameter that determines the effectiveness of biosurfactants
in various formulations and strongly determines the stability of emul­
sion [29–31]. In a surfactant molecule, the relative abundance of hy­
drophilic to lipophilic regions is shown by the HLB number - an arbitrary
scale of 0–20 [29]. By comparing biosurfactants with surfactants of
known HLB values/properties, the nature of emulsions formed by such
biosurfactants could be easily ascertained [32]. With regards to the
Bancroft rule, the HLB values of <6 (oil soluble) should be used in
water-in-oil emulsion (w/o) while emulsifiers with HLB values >10
(water-soluble) should favour oil-in-water (o/w) emulsion [32–36].
In complex formulations one vital parameter for the elucidation and
comparison of hydrophilicity and hydrophobicity of biosurfactants, as
noted by Jahan et al. [37], is the characteristic curvature value (Cc). The
Cc value – which depends on several factors (e.g., temperature,
hydrophilic-lipophilic deviation, salinity, etc.) – ranges from positive to
negative denoting “hydrophobic” and “hydrophilic” orientation,
respectively [37,38]. The Cc value could also be used to describe the
nature of micelle formation when surfactants are in solution. The sur­
factants with positive Cc value form reverse micelles (W/O micro­
emulsion) whereas negative values denote normal micelles (O/W
microemulsions) [38]. For instance, lactone-acetylated SL and RL,
owing to their Cc values, are comparatively hydrophobic and hydro­
philic, respectively [38]. Whereas the popular Griffin’s HLB number is
only useful for the non-ionic surfactants, it cannot be used for deter­
mining the strength of ionic surfactants. However, recent advances such
as the use of phase inversion temperature technique have been made
recently to assess a wider range of surfactants [39–41]. These properties
essentially determine the quality of a biosurfactant and depend largely
on the nature of the substrate used in the production and the prevailing
environmental conditions such as pH, temperature and ionic strength
[37]. The basic structural and physicochemical properties of the major
groups of industrially-relevant MGLs are summarized in Table 1.
Apart from the known classes of MGLs, novel members have also
been reported. For instance, MGL from non-pathogenic marine yeast –
Cyberlindnera saturnus SBPN-27 – has been described as cybersan (tri­
galactomargarate or Gal-Gal-Gal-Heptadecanoic acid) with ST = 28 mN
m− 1, CMC=30 mg L− 1 [26]. Nocardiopsis lucentensis MSA04 yields
another form of MGL with hydrophilic sugar, 3-acetyl 2,5-dimethylfuran
and hydrophobic non-polar hydrocarbon chain (nonanoic acid methyl
ester) [73]. Under nitrogen limitation, Rhodotorula babjevae Y-SL7,
(oleaginous yeast) synthesized an unusual MGL (a mixture of polyol
esters of fatty acid) [74]. Balan et al. [75] also showed that
marine-borne bacterium – Staphylococcus saprophyticus SBPS-15 – when
grown on glucose as a carbon source, secreted mannose-mannose-oleic
acid (staphylosan) which exhibited ST and CMC of 30.9 mN m− 1 and
24 mg L− 1, respectively.
3. Challenges of MGL production and role of process economics
in mitigation
The global surfactant market has witnessed an upsurge due to the
rising demand for surfactants or their allied products in the past decade.
For instance, the synthetic surfactants in the global market were esti­
mated at $30.64 billion in 2016 and projected to reach $39.86 billion in
2021 [76]. Also, the market for the biosurfactants estimated at $3.99
billion in 2016 was projected to surpass $7.95 billion in 2026 (Fig. 1).
Although a wide difference exists in the market value of the two cate­
gories of metabolites, biosurfactants – owing to their unique attributes:
eco-compatibility, biodegradability, mildly toxicity, and sustainability –
has continued to attract more attention in the past decade. For instance,
while the compound annual growth rate (CAGR) was at 4.3 %, in 2014
[81], it is currently projected to 6.9 % (this rate is expected to continue
C.O. Onwosi et al. Process Biochemistry 100 (2021) 124–139

Table 1
Physical and chemical properties of major MGL classes.
#
Chemical structure Producing organisms pH Surface tension reduction ▾
CMC Δ
Interfacial activity(mN/ ▴
HLB
stability (mN/M) (mg/L) M)

Rhamnolipids †
P. aeruginosa [43,44] 5–7 [48] 29 [43] 100 [50]
†
P. nitroreducens [45,46] 18.02 [44]

22–24 [51]
HXD:4 [51] 10.175
4–10 [47] 105 [49]
Lysinibacillus sphaericus [47] 32.21 [49] [52]

Xylolipids Lactobacillus helveticus [53] – 39.5 [53] 2.5 [53]

Lactococcus lactis [42]

– –
– 35.9 [54] 1.0 [54]
Pichia caribbica [54]

Trehalolipids 2–10 [55] 29 [56] 250 [55]

†
Rhodococcus erythropolis [55,
HPT: 4 [57] 9 [57]
56] 2–10 [56] 24 [57] 120 [58]

Mannosylerythritol lipids▀ Pseudozyma hubeiensis [59]

*Ustilago hordei [60]

1 1
4–10 [61] 30 dyne cm− [61] 15.8 μM [61] KER: 0.1 dyne cm− [61] 8.8 [61]
Candida antarctica [61]

Sympodiomycopsis paphiopedili
Cellobiose lipids▀▀
[62]
†
Cryptococcus humicola [63,64]

4.1(10− 4) M
– 37 [63] HXD: 10.0 [63] –
[64]
Trichosporon porosum [65]

Sophorolipids Starmerella bombicola [66,67] 32–43 [69]

Candida batistae [68]
Wickerhamiella domercqiae [69,
70]
2–10 [71] 20–130 [69] – 8–10 [72]
32.6 [71]

Rhodotorula bogoriensis [68]

#
(All the structures were adapted from [ref. 14] except xylolipid [ref 42], with kind permission from Elsevier).
†
Human pathogen; *Plant pathogen; ▾CMC Critical micelle concentration; ▴HLB Hydrophilic-lipiphilic balance; ΔHXDHexadecane; HPT Heptane; DHX Deca­
hexane; KER Kerosene ▀MEL (R1=Acyl (C2-8); R2=Acyl (C12 - 20); R3, R4=H or acetyl) ▀▀ CL (CL A: R1, R3, R4=H; CL B: R1=H, R3=a, R4=b; CL B: R1=CH3, R3=a,
R4=b, R2=OH or H).

until 2026) [80]. The major factors driving the current market growth
are the growing demand for green products due to rising consumer
awareness of the excellent benefits of the biobased metabolites,
increasing ecological awareness, affordable fermentation feedstocks,
lower carbon footprint and favourable government regulations [80,82].
Although the literature is replete with reports describing interesting
outcomes from different investigations on MGLs production, replicating
them at commercial scale continues to be complicated, unpredictable
and costly [83]. As observed in other biotechnological products, the
process economics is a major drawback to the commercialization of
MGLs production [20,78,84,85]. For instance, while the current price of
low-cost MGLs (e.g., SL) is reported $34/kg active matter, the prices of
synthetic surfactants such as amino-based specialty surfactants and so­
dium lauryl sulphate (bulk application based) fall in the range of
$3–4 kg− 1 and $1–2 kg− 1, respectively [22,86]. Thus, it can be deduced
that the price of generally accessible MGLs (SL) is approximately 10–15
times higher than its chemical counterparts. This underlines the need to
reduce cost of production of MGLs to make them commercially attrac­
tive in personal care and biomedical applications [22]. Diverse factors
are responsible for the high cost of MGLs production at commercial scale
[87]. One of the major reasons is that the fermentative media for the
growth of MGL-producing microorganisms is chemically synthesized
[83,88]. From an industrial viewpoint, this creates a considerable
financial variation between investment and realizable productivity [83].
Another hindrance is that the bioprocess has low efficiency. The issues of
low yields (low titer and volumetric productivity) have led to higher
prices of biosurfactants, making them less competitive with synthetic
surfactants in the global surfactant market [20,83,87,88].
To make MGLs production commercially feasible, it is important to
understand the underlying process economics. In every production

126
C.O. Onwosi et al.
Fig. 1. Global biosurfactants market value and size projections from 2015
through 2026.
(Data source: ref. [77–80]).
process, especially at the industrial scale, there is a relationship between
the input (capital and labour) and output [89]. The long-run average
cost (LRAC) curve (also known as planning curve) shows the functional
relationship between LRAC of production and the output, by assuming
that all production factors are variable (Fig. 2). From the illustration
(Fig. 2), it could be deduced that LRAC curve shares an inverse rela­
tionship with the return to scale. On the one hand, firms experience
diminishing LRAC and increased economies of scale (i.e., increased
returns to scale or more output at lower cost). On the other hand, firms
are faced with diseconomies of scale when there is a rising cost of pro­
duction (decreasing returns to scale or more output at a higher cost)
[90]. Considering that all costs can be varied in the long run, the firm
can consider the alternative strategies that would result in the lowest
cost of production [91]. Therefore, firms involved in industrial MGLs
production could rely on the economies of scale to achieve a lower cost
of unit production. Operation cost associated with MGL production
Fig. 2. A schematic of long run average cost curve of large-scale glycolipid
production.
Stages I, II and III refer to laboratory, pilot and commercial scale, respectively.
In the long run, a firm operating at commercial scale experiences three sce­
narios: economies of scale, constant returns to scale and diseconomies of scale.
Economies of scale refers to a situation where output increases (QX to QY) as
the average cost decreases (CX to CY). At Constant returns to scale, expanding
all inputs proportionately does not change the average cost of production (point
CYQY). Diseconomies of scale – the long-run average cost of producing each
unit increases (CX to CZ) as total output increases (QX to QZ). Therefore, the firm
maximizes profit by reducing the cost of production at the barest minimum (CX)
and maintaining the level of output below QY. This can be achieved by using
low-cost feedstock, the use of integrated and affordable downstream processing,
merging with other firms to increase the volume of output, etc. However, it is
important to note that market demand also has a significant role in determining
the output of a product.
127
Process Biochemistry 100 (2021) 124–139
process at commercial scale can be appreciably minimized by using
low-cost or cost-free agro-industrial waste (e.g., food wastes, agricul­
tural by-products, oily wastes) as feedstock [83,92]. Increasing the size
of MGL-producing firms via merging of the existing firms or acquisition
of budding firms by already established ones could increase the opera­
tional volume with the resultant decrease in the unit cost of production.
Deploying affordable in situ separation technologies (downstream pro­
cessing) is also a fundamental economic approach for successful
advancement to large-scale MGLs production [93]. However, the cost of
production is not the only driving force for the increased output of a
particular product; the market demand also plays a significant role.
Thus, to experience improved returns to scale, firms engaged in MGLs
production should adopt strategies that can, not only reduce the cost of
production but also present high-quality products to the competitive
surfactant market. Apart from cost reduction strategies, firms can
improve their market competitiveness through product differentiation
(non-pricing differentiation) and focused production (e.g., use of
renewable wastes for MGLs production could help control climate
change).
4. Competitive marketing strategies adopted by various MGL-
producing firms
The main reason for the industrial production of any commodity is
profit maximization from a minimum investment [83]. Thus, firms
producing these biometabolites at commercial scale are saddled with the
responsibility of formulating a competitive production and marketing
strategy that would ensure their product has an edge in the market while
being also profitable. Interestingly, MGLs with unique blends, which
have been developed to meet various consumers’ demands in biomed­
ical and personal care, are in supply from different companies world­
wide (Table 2). To make MGLs attractive in the surfactant market, these
companies operating at varying capacities have developed strategies
that make their products competitive. In this section, we will be
addressing some of these techniques.
According to Slater and Narver [114], there exists a positive rela­
tionship between a business operation size and its performance. Inter­
estingly, some big biosurfactant producing companies are forming
mergers. Evonik Specialty Chemical Company based headquartered in
Essen, Germany, is one of the foremost chemical companies to utilize
biotechnological protocols to produce MGLs – SL and RL – relevant in
personal care products at commercial scale [97]. The uniqueness of
Evonik’s MGLs products is the flexibility of the foaming properties as
they can be modified for each application. Thus, it’s one of the most
sought-after products in the formulation of shower gels and shampoos
[97]. To further strengthen its stand in the industrial scale MGLs pro­
duction, Evonik has formed a merger with Unilever (renowned for sus­
tainable beauty and personal care products development) to improve the
output of the “green products”. An interesting outcome of this partner­
ship is that products can be made at a reasonably low cost [115]. The
acquisition of budding firms by larger and more established firms has
been reported. For instance, acquisition of Logos Technologies, USA
(that developed NatSurFact® RL) by Stepan Company, USA, to
commercialize product to meet consumers’ demand [108]. Givaudan,
Switzerland (maker of fragrance and flavour) acquired Soliance, France
(cosmetic ingredient firm). With Soliance playing an active role with its
advanced technologies, the acquisition was made to strengthen Givau­
dan’s process and research capabilities in the development of MGLs at
commercial scale [96].
Large companies can also take advantage of joint research with
universities or research centres. This partnership reduces research ex­
penses and lowers the risk associated with nonproductive outcomes as
biotechnological firms, most times, are involved with a considerable
degree of investment with no guarantee of success. Therefore, innova­
tive research leading to the production of unique MGLs can only be
undertaken by companies with significant resources or by partnering
C.O. Onwosi et al. Process Biochemistry 100 (2021) 124–139

Table 2
List of MGL companies with interest in biomedical and personal care products.
S/N Company Product Registered trademark Purity (%) Price Area of interest Ref.

1. MG Intobio Co. Ltd., South Korea SL Sopholine® NA NA Anti-acne soap, face mask. [94]
2. Allied Carbon Solutions, Japan & USA SL ACS-Sophor® NA NA Hair care, anti-acne, antimicrobials [95]
3. Givaudan, Zurich, Switzerland SL Sopholiance® S NA NA Antibacterial, anti-acne [96]
4. Evonik Specialty Chemicals, Germany SL, RHL NA NA NA Personal care (shampoo, shower gel) [97]
5. Holiferm Technology, UK SL, MEL HoneySurf ® LF/HF NA NA Personal care [98]
6. Toyobo Technology, Japan MEL SurfMellow® NA NA Skin care/ moisturizer [99]
7. Kanebo Cosmetics and AIST, Japan MEL Twany Seasonal Essence® NA NA Personal care (skin moisturizer) [100]
8. Biotopia Co. Ltd., South Korea MEL NA NA NA Personal care [101]
9. Fraunhofer IGB, Germany. CL, MEL NA NA NA Emulsifier, cosmetics [102]
10. RL* AGAE Technologies RHL RL* AGAE 90–95 1250/kg Cosmetics, oral care, pharmaceutical [103]
11. Sigma Aldrich, Germany RHL NA 90–95 $3253/kg Emulsifier 104,105]
12. Jeneil Biotech Inc. RHL NA NA NA Personal care, antimicrobials [106]
13. Paradigm Biomedical, Inc., USA RHL NA NA NA Antimicrobials, wound healing [107]
14. Stepan Company Inc., USA RHL NatSurFact ® 85–90 $700/kg Antimicrobials, wound healing 105,108]
15. Urumqi Unite Bio-Technology Co., Ltd. RHL NA NA NA NA [109]
16. Daqing Victex Chemical Co., Ltd. RHL Rhamnolipid B25 NA NA Personal care [110]
17. Glycosurf Inc., USA RHL NA 95–98 $525/kg Personal care, medical device cleaning [111]
18. Biotensidon GmBH, Germany RHL Rhapynal® NA NA Cosmetics, pharmaceutical [112]
19. TENSIOGREEN Technology Corp RHL NA 90 NA Personal care, biomedical [113]

SL – Sophorolipids CL – Cellobiose lipids MEL – Mannosyl erythritol lipids RHL – Rhamnolipids NA – Not available.

with research centres. For instance, Toyobo Co. Ltd. (Japan), in
collaboration with AIST (Japan), has developed a skin moisturizing
lotion – SurfMellow® – that has a ceramide-like MELs synthesized by
Pseudozyma tsukubaensis as the active ingredient. At a very low con­
centration (0.1 %), this product restored moisture content of SDS-
damaged model skin [99]. Also, in conjunction with the AIST, Japan,
The Kanebo Cosmetics, Japan, has developed a four-week skincare
formulation - Twany Seasonal Essence – specifically made for effective
recovery of damaged skin resulting from an irregular structure, rough­
ness and dryness. This formulation comprises vitamins, sodium surfactin
and mannosylerythitol lipid B (MEL-B). The strong surface activity and
elevated moisturizing activity of MEL-B makes it an active ingredient.
The sustainable production of MEL-B using cheap raw materials could
improve the competitiveness of the products in the surfactant market
[100].
Another important approach used by large firms benefiting from
lower average cost of production is economies of scope. It occurs when a
large firm utilizes its existing resources to diversify into related markets
[116]. This diversification could involve the production of allied prod­
ucts or by the production of helpful service. The majority of the
MGL-producing firms such as Jeneil Biosurf Inc and Evonik have also
engaged in the production of other useful metabolites. Holiferm Tech­
nology, an emerging MGL-producing company, provides technical ser­
vices to other MGLs firms [98]. In the commercialization of
biotechnological products, the two most economically key performance
indicators (KPIs) the product titers and volumetric productivities.
Holiferm Technology has reportedly advanced these KPIs for SL pro­
duction – product titers >800 g L–1 and volumetric productivities
>5 g L–1 h–1 – and substantially reduced the cost of production. This has
been demonstrated at pilot scales at different commercial sites and the
process economics and sustainability have been verified for SL produc­
tion. Interestingly, this concept has also been shown to be feasible for
commercial production of MEL and RL [98].
Several MGL-producing firms use other strategies such as product
differentiation to make their products unique. This type of product dif­
ferentiation is called the non-pricing differentiation since it is based
solely on product quality. For instance, Biotensidon GmBH, a Germany-
based biotech company, has commenced mass production of unique
forms of RLs. With RL output of 200 tons at the commencement of
production in 2015, the company has recorded an estimated output of
5000 tons in 2019 [112]. The Biotensidon RL brand (Rhapynal®) is an
effective, pro-health and environmentally compatible supramolecular
multiplex that comprises: RL (surface active agent), pyoverdines (fluo­
rescent siderophore) and alginates (network forming carrier matrix).
The product is sustainable, innovative, biobased and comes in as two
variants – Rhapynal®-powder and Rhapynal®-liquid – to meet varying
demands from end-users. Rhapynal® – being a complex formulation – is
multifunctional with antiviral, antifungal and bacterial properties. It
also acts as a biological booster and emulsifier in biomedical and
cosmetic product formulation because it considerably minimizes the
numberof active ingredients required for the design of a product [112].
Sopholiance® S (SL biosurfactant) currently owned by Givaudan,
Switzerland, is used in a wide range of cosmetic products (acne-prone
skincare, facial cleansers, oily skin products, deodorants and make-up
removers [96,117]. Specifically designed for cosmetic manufacturing
and ready-to-use cosmetic emulsions, Sopholiance® S is very effective at
low concentrations (0.5–3%) at temperature <40 ◦ C. As an active
ingredient in cosmetic products formulation with antibacterial proper­
ties, it specifically targets and suppresses the causative agents of acne –
Propionibacterium acne. It has been reportedly used by different cosmetic
agents (e.g., Cattier, Melvita, Naturopathica, Bioderma, etc.) [117].
Finally, the majority of the MGLs-producing firms have remained
competitive by adopting the focus strategy – a competitive strategy
where the firms focus attention on a market segment where the company
can compete favourably. For instance, targeting the global interests in
environmental sustainability, lower carbon footprint, and ultimately,
mitigation of climate change, MGLs are being presented in the market as
biocompatible “green” products.
5. R & D towards commercialization of MGL
The relatively low volumetric productivity and high cost of MGL
production remain the major challenges stalling their commercializa­
tion. For MGLs to find good application in cosmetic and biomedical
applications, consolidation should be made on replicating the
laboratory-based investigations at a commercial scale. Herein, we pre­
sent the several biotechnological advances made by investigators to­
wards reducing the cost of production, improving yield and overcoming
biosafety concerns due to pathogenic strains.
5.1. Genetic engineering targeted at creating platform organisms
Genetic alteration is often targeted at developing platform organisms
with enhanced robustness for enhanced MGL production [118]. For
example, to circumvent the challenge of pathogenicity of the traditional
RL producing strain, Pseudomonas aeruginosa, heterologous expression of
rhlABRI gene cluster in non-pathogenic organisms such as P. putida
[119], P. stutzeri [120] and P. chlororaphis [121]. This important

128
C.O. Onwosi et al.
approach could reduce the cost of production of RL since using the
pathogenic organisms requires extra purification steps due to pathoge­
nicity concerns. However, the demerit of this protocol the MGL pro­
ductivity and titer of the non-pathogenic organisms are low. Therefore,
modifying the natural pathogenic MGL producing strains could be a
more promising strategy for improving MGL production [122]. For
instance, considering that pyocyanin demethylase (PodA) inactivates
the pyocyanin (virulence factor) by converting the methyl group to
formaldehyde, the introduction of PodA into the fermentation broth has
been suggested as a potential method of mitigating the pathogenicity
concerns when deploying P. aeruginosa MGL production [16].
According to Yang et al. [123], low MGL yield stems from difficulties
in selecting the essential metabolic loci necessary for the overproduction
of MGLs. Therefore, metabolic fine-tuning is an essential technique to­
wards the production of novel biosurfactants tailor-made for unique
applications. Sodagari and Ju [124] noted that exhaustion of nitrogen
source during RL production by P. aeruginosa slowed down or stopped
the fermentation process, irrespective of the concentration of cells in the
broth. They demonstrated that the partial replenishing of broth with
media containing N-source reactivated the process. It was concluded
that extending the stationary phase, the labour and downtime for the
initiation of short intermittent batches will be averted. Also, the feed­
stock and time consumed for growing cells will lessen. This approach
enhances the process economics of RL as it reduces cost, improves the
product yield as well as the volumetric productivity. Roelants et al. [19]
have underscored the suitability of SLs in personal care and biomedical
applications. The authors noted that the enzyme – lactone esterase from
Starmerella bombicola – involved in lactonization of acidic SL could
improve the specificity of pure forms of the final product (about 98 %
pure acidic or lactonic SL). Yang et al. [123] also demonstrated that
optimization of the citrate feeding protocol as well as harnessing the
other carefully manipulated genetic protocols (e.g., improved SL trans­
port by overexpression of mdr gene, improved ATP:citrate lyase activity,
metabolic balance analysis, etc.) enhances the yield and titer of SL.
5.2. In silico computation for discovery of novel MGL metabolic pathways
To optimize RL production, Pseudomonas putida expressing RL genes
(rhlAB) from P. aeruginosa has been constructed using the in silico (ma­
chine learning along with multi-omic modelling) engineering technique
[125]. According to Occhipinti et al. [125], this protocol provided
computational structure to fuse multi-omic data towards the identifi­
cation of untapped genes and pathways for enhanced production of RL
in the chassis organism (P. putida). Currently different online databases
are available for mining information on proteins specific data, calcula­
tion of molecular properties and pathways relevant to in silico engi­
neering. These include BRENDA (BRaunschweig ENzyme DAtabase),
FRENDA (Full Reference Enzyme DAta), AMENDA (Automatic Mining of
Enzyme DAta), and KEGG (Kyoto Encyclopaedia of Genes and Genome)
[126,127]. Other in silico computational techniques used for forecasting
metabolic intervention (revealing novel genes and pathways) or
improving the targeted biometabolite overproduction include Ideal­
Knock, OptKnock and OptForce algorithms [127,128].
5.3. Use of sustainable and renewable biomass for MGL production
Especially with the increased interest in reducing environmental
impacts and climate change due to anthropogenic activities, research on
exploitation or refining of biowastes as raw materials – bio-based
economy – for the sustainable production of biopolymers, biofuels and
biochemicals have received growing attention in the past decade [129].
By developing a process model for the economic assessment of
fermentative production of MGL, Ashby et al. [130] pointed out that the
cost of raw materials (feedstock) constituted about 87–89% of the
overall operational cost. As raw materials are the major contributors to
the overall operational cost of MGL production at a commercial scale,
129
Process Biochemistry 100 (2021) 124–139
using a low-cost feedstock derived from agro-industrial processes will be
beneficial to any potential investor [83]. However, to select low-cost
materials as fermentative media for biosurfactant production, it is
essential to take into account some factors such as a sustainable source
of the material, a requirement for pretreatment, cost of transportation,
need for the addition of extra raw materials, etc [92]. According to
Sekhon Randhawa and Rahman [85], the prices of crude and pure
glycerol are at the range of $0.04 – $0.33/kg and $0.50 – $1.50/lb,
respectively. The price is expected to drop given the renewed interest in
sustainable and renewable energy such as biodiesel which is the major
supplier of glycerol (100 kg of glycerol is generated from the production
of 1 metric ton of biodiesel) [85]. The prices in USD of sustainable
feedstocks such as cane bagasse, rice husks, corn steep liquor are
$0.04/kg, $0.08/kg and $0.46/kg, respectively [131]. These prices are
known to be 1–2 folds lower than the prices of locally available chem­
ically defined raw materials (e.g., glucose, sucrose, etc.) used in MGLS
production [92].
By fermenting canola frying oil with Pseudomonas cepacia in 50 L
bioreactor, Soares da Silva et al. [132] recovered MGL (40.5 g L–1)
estimated at USD $20/kg. Replacing the mainstream carbon source (e.g.,
glucose) with renewable lignocellulosics – wood hydrolysate – during RL
production by P. aeruginosa has been reported to be a cost-effective
approach [133]. Other low-cost carbon substrates successfully utilized
in RL production include palm fatty acid distillate [43], based Distillers
Dried Grains with Solubles [134], rice-straw hydrolysate (C6 stream)
[135]. To reduce the cost incurred during SLs production, different
biowastes (hydrophobic and hydrophilic substrates) have been used as
sustainable and cheap carbon sources. Maddikeri et al. [136] utilized
ultrasound pretreated waste cooking oil and recorded >120 % increase
in lactonic SL yield during a fed-batch operation. Konishi et al. [137]
used corncob hydrolysate – a lignocellulosic-derived feedstock – for SL
production and reported volumetric productivity of 12.3 g L− 1d− 1 (i.e.,
0.51 g L− 1 h− 1). Food waste valorization has had great attention given
the sustainable development plans of environmental preservation, mass
and energy balances as well as food security. Overall volumetric pro­
ductivity of 1.25 g L− 1 h-1 was achieved by using food waste hydrolysate
[138]. However, for MGL-forming microorganisms to effectively utilize
lignocellulosic hydrolysate, they must be metabolically competent to
consume the sugars such as xylose (product of hydrolysis of hemicellu­
lose). Using in silico metabolic network framework and flux balance
analysis, Bator et al. [139] created a platform organism – P. putida
KT2440 – by heterologous expression of different xylose utilization
pathways (e.g., Dahms, Weimberg and isomerase). They noted, after
adaptive evolution, that the chassis organism effectively utilized xylose
for RL production.
Apart from the reduction in production cost, the application of low-
cost raw materials in MGLs production at an industrial scale has other
merits such as sustainability, eco-compatibility and improved yield/
productivity. However, it should be relevant to underline that affordable
raw materials could present several shortcomings. These include the
presence of impurities in the final product, presence of undesirable
components in the substrate, requirement for extra pretreatment steps to
access the essential nutrients, may lack uniformity and could result in
increased production cost [83]. Therefore, the utilization of low-cost
feedstocks must be carefully managed and could require further in­
vestigations and cost calculations as they sometimes result in poor
product yield [68].
5.4. Bioprospecting of hyperproducing strains using high throughput
screening
Another important approach for reduction of cost of MGL production
or its commercialization is the search for novel MGL-producing micro­
organisms (both culturable and unculturable), using robust screening
procedures. Traditional methods have long been described for MGL
production – the primary (drop collapse, oil spread) and secondary
C.O. Onwosi et al.
(haemolytic assay, cetyl trimethyl ammonium bromide agar test,
emulsification index (E24), emulsification activity, surface tension)
screening [73,140,141]. However, these approaches are generally
laborious, time-consuming, qualitative or semi-qualitative and inca­
pable of handling large numbers of isolates. On the other hand, the
discovery of novel MGL-producing organisms from several isolates re­
quires robust detection methods, such as high throughput screening
(HTS) performed mainly on microplates [142]. They are usually quick,
reliable, quantitative and easily compared. A HTS – semi-quantitative
atomized oil assay – which requires several serial dilutions as well as
observation under a microscope has been described [142].
While no new idea has been established by HTS, its miniaturization
as well as automation has undoubtedly aided the evaluation of several
potential biosurfactant-producing strains [143]. HTS also helps to
identify intermediate phenotypes (mutants) [142]. It is also advanta­
geous as it has broader applicability and detects biosurfactants at much
lower concentrations (about 10-fold) compared with the conventional
approaches [142]. Another source of interest in this technique is that it
helps ascertain the HLB categories of biosurfactants. For instance, it has
been noted that low (8–10) and high (>13) HLB surfactants showed
bright and dark halos, respectively, in the course of the assay [142]. HTS
has also been extended to the functional screening of metagenomic li­
braries through the construction of expression systems [144]. This
approach aids in the detection of novel nonculturable
biosurfactant-producing isolates [144,145]. Molecular detection and
quantification of functional biosurfactant-producing communities could
be done with DNA microarray, stable isotope probing and fluorescence
in situ hybridization assays [145]. While the majority of the screening
techniques involve the use of culturable microbes, it is thought that a
vast number of unculturable organisms could harbour rich potentials for
MGLs production. Consequently, functional metagenomics has been
deployed as a robust tool towards discovering novel MGL from uncul­
turable organisms. Since this approach does not require PCR, it offers
numerous advantages such as exploring clusters of genes encoding
pathways relevant to biosurfactant production [145].
5.5. Downstream processing of MGLs
According to Mukherjee et al. [20], the costs associated with
downstream protocols constitute about ~60 % of the total operational
cost of production of biotechnological important products such as bio­
surfactants. While purity level and substrate’s source do not necessarily
affect the application of MGLs in environmental remediation, their
high-value-added use (e.g., pharmaceutical adjuncts, wound healing
and cosmetic formulations) requires a higher level of purity level and
specific origin of the feedstock [146,147]. Especially in scenarios where
the purity of the product is paramount such as biomedical and personal
care products, the downstream processing – which requires extensive
purification steps – constitutes 70–80 % of the overall operational costs
[85]. This could be attributed to the requirement for large fermenter
volume due to low productivities during the downstream processing of
MGLs [148]. Therefore, despite the increased interest in biosurfactants,
their bulk utilization has not been realized due to unfavorable prices
especially when they are compared with chemical surfactants [148]. For
industrial-scale production of any commodity scale biometabolite such
as MGLs to be feasible and profitable, a volumetric productivity
benchmark of ≥ 2 g l− 1 h− 1 has been suggested [22,148]. However, the
majority of the MGLs have not reached this threshold at
commercial-scale production due to the use of conventional (chemical)
treatments during the recovery process. Utilization of traditional pro­
tocols (e.g. solvent extraction, distillation) for the recovery of bulk
chemicals often results in irreversible damage to the producing cells
[146]. Therefore, it requires several rounds of startup, sterilization and
general cleaning, hence, making the MGLs production process uneco­
nomical [148].
Hence, to avoid this outcome, the producing cells are first recovered
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Process Biochemistry 100 (2021) 124–139
before the products are harvested. These extensive protocols lead to
multiple startups of batch processes, cleaning and sterilization, loss of
operational time; low product yields and ultimately makes the cost of
production very prohibitive. An in situ integrated (gravity-based) sep­
aration protocol that allows for simultaneous production and harvesting
of MGLs has been developed to avert these drawbacks [149]. For
instance, Dolman et al. [146] reported a fermentative production of SL
that lasted for 1000 h yielding 623 g on an initial working volume of 1 L
(i.e., volumetric productivity of 0.62 g L− 1 h − 1). According to Dolman
et al. [148], integrated product recovery techniques improve the pro­
ductivities and titers. Especially for SL recovery, this approach offers
two main merits: a) It helps to control oxygen limitation resulting from
high viscosity SL produced during the fermentation process; b) It pre­
vents feedback product inhibition of the fermentation process since high
concentration of SL suppresses the genes (ugtA1 and ugtB1) essential for
their biosynthesis [147]. One major shortcoming encountered, herein, is
the loss of substrate (oil/oleic acid) during separation. Therefore, in the
course of developing a separator, precaution should be taken to mini­
mize biomass and nutrient loss during the separation protocols. To avoid
the substrate loss encountered by Dolman et al. [146], Zhang et al. [150]
deployed a two-stage separation system (dual ventilation pipes and dual
sieve-plates) to recover oil resulting in about 61 % improvement
(volumetric productivity of 1.59 g L− 1 h − 1 under 300 h fermentation).
While the latter report improved the SL purity and yield, the separation
approach required complex fabrications. It is noteworthy that this
complex system may increase the chances of contamination and could
result in higher production costs. To prevent the production hitches
emanating from SL build-up during fed-batch fermentation, Liu et al.
[67] realized overall productivity of 1.55 g L− 1 h− 1 via applying froth
floatation technology as an in-situ separation technique. Utilizing the
fed-batch fermentation of restaurant leftover hydrolysate and simple
in-situ separation, Wang et al. [147] achieved 1719.7 g from 1.5-L
operational volume during 480-h fermentation (i.e., volumetric pro­
ductivity of 2.4 g L− 1 h− 1). However, the proposed technology could be
of importance when implemented industrially due to its numerous at­
tributes: high recovery rate, rapid, ensures product safety (e.g., it re­
duces the chances of contamination), prevents loss of cell biomass,
supports higher operational volume and ultimately reduces the pro­
duction cost (i.e., it does not require additional chemicals for the re­
covery of the product, thereby saving the cost of further purification
steps).
Other robust integrated separation techniques such as foam frac­
tionation and membrane separations have been reported to be indis­
pensable for the recovery of MGLs in general [148]. For instance, one of
the major issues with RL production at the laboratory scale is foam
formation [151]. It has discouraged continuous fermentation as a result
of low volumetric productivity. Therefore, attempting to replicate the
laboratory protocols at the industrial scale requires adequate technolo­
gies to manage foam build up [151]. According to Jiang et al. [151], the
use of in situ foam fractionation and membrane filtration could be reli­
able options for foam destabilization under large scale RL production.
They further suggested integrating different foam control strategies such
as chemical and mechanical breakers, stop valves, etc. Adopting
sequential fed-batch fermentation would also help destabilize foam
formation issues during RL production. These foam fractionation tech­
niques would effectively enhance the operational volume and make
large-scale production of RLs economically feasible.
Taken together, the robust integrated separation techniques (foam
fractionation, membrane and gravity separations), which extend the
period of fermentation processes, enhance titers and volumetric pro­
ductivity during MGLs production [148]. They are affordable, simple,
reduce the fermenter volume required for production and lower the
number of intermittent batches.
Interestingly, recovery procedure such as in situ gravity separation
has been successfully assessed at the pilot scale (30 L working volume)
and also shows promises to be replicated in 200 L bioreactors [147,149].
C.O. Onwosi et al.
This marks a significant milestone given that translation of many other
lab-scale integrated separation protocols to a large scale has not been
realistic [147].
5.6. Optimization of vital fermentation conditions
Some of the modeling approaches include the response surface
methodology [152], artificial neural network (ANN), etc. Interestingly,
quantitative structure–property relationship approach has also been
used to develop clusters of equations that could be indispensable in the
biosynthesis of RL biosurfactants for specific applications. Using this
modified evolutionary algorithm, a relationship between CMC, pH, hy­
drophobicity (log Kow) and purity of RL was described [153]. Using this
model, they noted that CMC of RL depends on the log Kow of the carbon
source used in its biosynthesis and less pure RL showed higher CMC. This
could be considered as a viable option in the optimization of functions of
biosurfactants for cosmetic and health care applications.
6. Techno-economic assessment for sustainable
commercialization of MGL production
At present, the commercialization of MGL production is economi­
cally challenging due to high cost of feedstock and downstream pro­
cessing. Techno-economic assessment (TEA) is an indispensable
decision-making model for evaluating the potential economic viability
and technological options necessary for the commercialization of a
production process [154]. It is a foundational tool that provides insights
into alternative production pathways essential for future commercial
development of products, especially where no previous information on
industrial facilities is available [154,155]. By linking financial evalua­
tion and economic approximations (often derived from laboratory-based
studies data performed under short duration and highly-controlled
conditions) with process modeling (performed in simulation software),
TEA estimates the cost benchmarks of products [154]. Thus, cash flow
delineated by TEA (especially before a new project is launched) ensures
that technological risks associated with the commercialization of a given
product are minimized [156]. Therefore, the precision of TEA involves
significant technical awareness of the process, to adequately represent
the mass and energy balance estimations on process-unit-level rationale
[154,157,158]. In general, the TEA framework comprises four major
steps as shown in Table 3.
Although TEA helps minimize the risk associated with the early
stages of technological systems, translation of laboratory-scale data to
commercial scale is confronted with many bottlenecks (uncertainties).
The effects of different uncertain parameters such as feedstock price,
metabolite price, capital cost, maintenance rate, interest rate, etc., are
often neglected in TEA of metabolite production [156]. While the
identification and modeling of numerous production pathways have
been realized, the variability in processing assumptions and modeling
boundaries has resulted in uncertainties in current scalability results
[154]. The uncertainties could be process inherent such as inconsistency
in feedstock supply, system performance and product yield [163].
Considering that the number and level of detail for each variable posi­
tively influences the prediction of the selling price of a commodity, TEA
(with single NPV) does not sufficiently reveal economic risk related to
the selling price. Thus, an uncertainty analysis is important for
improving the accuracy of results presented in TEA. Different stochastic
and deterministic approaches have been used for uncertainty evalua­
tions: scenario and sensitivity analysis; Global sensitivity analysis and
Monte Carlo; use of pedigree matrices in the qualitative estimation of
quantitative uncertainty values [163–165]. Another approach such as a
non-probabilistic strategy which employs a non-probabilistic reliability
index has been proposed as a reliable tool for uncertainty analysis [166].
Thus the uncertainty evaluation result serves as an indispensable guide
to identify and differentiate important parameters from the unimportant
ones [156]. This approach would be helpful for improved TEA of MGL
131
Process Biochemistry 100 (2021) 124–139
Table 3
General framework for TEA of MGLs production.
Step Description
1. Market Investigation of the market perspectives and external factors
study affecting the commercialization of MGL.
2. Simulation This involves the simulation of the MGL production plants: raw
description material processing capacity, location, longevity, procurement,
installation, operation mode and duration are performed using
software such as SuperPro Designer 8.0® or Aspen Plus®.
3. Process For technical feasibility, the MGLs production process being
description simulated is based on a lab-scale investigation. Herein, the process
flow is designed and the parameters for process development are
stated.
This is performed by estimating the following:
a) TCC = Equipment purchase cost + additional direct/indirect
cost + working capital cost
b) APC = CRM + CU + CL + CM + CLB + CMI
c) AR = Revenue accumulated over a particular period from the
sales of the product, by-products, and service fee paid by gov­
ernment for waste treatment.
d) Profitability analysis: This is performed by considering the
following:
i Minimum selling price (US$ MT–1) occurs when the net
present value (NPV) = 0
ii The unit production cost (US$ MT− 1) =
Annual production cost
Total glycolipid (SL, RL, XL, MEL or TL) production
4. Economic iii Gross profit = AR – APC
evaluation iv Net profit = AR – (APC + income tax or depreciation)
∑ Ct
v NPV(US$) = Tt − Co
(1 + d)t
Annual net profit
vi Return on investment (ROI), % = ×
Capital cost
100
vii Internal rate of return (IRR) = i1 + (i2 − i1 ) ×
NPV1
NPV1 + |NPV2 |
|CNCFT− 1 |
viii Payback period (Pt) = T − 1 +
NCFT
e) Sensitivity analysis: This is performed to assess the effect of
different parameters upon the economic performance. The
fluctuation in the economic environment during plant’s lifetime
is considered. The sensitivity analysis is performed using
Microsoft® Office Excel.
Adapted from ref. [159–162].
TCC = Total capital cost APC ¼ Annual production cost AR ¼ Annual revenue
CRM ¼ Cost of raw materials.
CU ¼ Cost of utilities CL ¼ Cost of labour CM ¼ Cost of maintenance CLB ¼ Cost
of laboratory CMI ¼ Miscellaneous NPV ¼ Net present value CO ¼ Initial in­
vestment t ¼ Plant lifetime (years).
Ct ¼ Net cash flow during t d ¼ Discount rate i1,i2 ¼ Basic discount rates.
T ¼ Year in which cumulative net cash is positive or zero for the first time NCFT
¼ Net cash flow in year T CNCFT-1 = Cumulative net cash flow in T-1 year.
commercialization.
Considering the framework of sustainability, factors that should be
considered in TEA are feedstock, social/environmental impact and en­
ergy utilization. Innovative protocols for MGL production should,
therefore, focus on the reduction of economic costs and environmental
footprints. Utilization of food wastes as feedstock is economical because
it would reduce environmental footprint (e.g., greenhouse gas emis­
sions) as well as the cost of production [161]. Evaluation of MGLs such
as sophorolipids and rhamnolipids showed that thermal requirements,
electricity and gas emissions are the main factors impacting the envi­
ronment [167]. Therefore, technology integration for MGL production
pathways should eliminate emission or energy-demanding protocols and
incorporate technologies that reuse or recycle waste streams to maxi­
mize output. The integration of MGL production in biorefinery has been
suggested as a beneficial step towards improving the current production
pathways, considering that TEA may result in very expensive or
 Table 4

C.O. Onwosi et al.

Summary of applications of MGLs in biomedicine and cosmetics.
Glycolipid sub-group

RL Cl TL Xl Mel SL

Skin care
[172] – [56] – [180] [184]
132

[173] – – – [181] [185]

– – – – [182] [186]
– – – – – [187]
– – – – – [5]
– – – – – [5]
Biomedical applications
Immunomodulatory [174] – [178] – – [188]
Antibacterial [175] [177] – [42] [183] [177]
Antifungal [175] [177] – – – [189]
Antiviral [176] – [179] – – [190]

Process Biochemistry 100 (2021) 124–139

SL – Sophorolipids CL – Cellobiose lipids MEL – Mannosyl erythritol lipids RHL – Rhamnolipids TL – Trehalolipid.
C.O. Onwosi et al. Process Biochemistry 100 (2021) 124–139

Fig. 3. Expression of α-smooth muscle actin (α-SMA) in di-rhamnolipid-treated fibroblasts and myofibroblasts.
The α-SMA expression is one of the major fibrotic indicators of myofibroblasts. The immunofluorescence staining and Westerern blotting assays reveal that α-SMA is
significantly suppressed in myofibroblasts by di-rhamnolipids. It is important to note that α-SMA is not remarkably expressed in fibroblasts. R10, R20 and R30 = di-
RL at concentration of 10, 20 and 30 mg/L, respectively. Fib = fibroblast, Myofib = myofibroblast, RHA = di-rhamnolipid (Adapted from ref. [169], https://www.nat
ure.com/articles/srep37553).

unsustainable processes [155,168]. With a more robust and integrated
plant, high-value metabolites could be produced alongside MGL;
thereby reducing the production unit cost and enhancing the profit­
ability of the overall process [155]. The higher sustainability provided
by biorefinery could permit better adaptability in regards to market
variability from feedstock, recycling protocols and production path­
ways. Given that economic feasibility and environmental footprints of a
process should be optimized, it has been suggested that life cycle
assessment (LCA) and resource assessments be integrated with TEA
[154,155]. However, the fact that these tools are separately determined
further complicates the comparisons of the novel technologies and
different production routes associated with TEA, LCA and resource
assessment [155]. Therefore, there is a need to provide uniform system
boundaries that simplify the comparison of processing techniques when
applied in TEA of MGL production
Resource availability and utilization should be integrated into TEA

Fig. 4. Gene delivery system by liposomes containing MGLs.

Novel liposomes developed by mixing the MGL with cationic
lipids) efficiently bind to DNA and aids gene transfer into cells.
The new method enables a 50–70-fold increase in the rate of
gene transfer into a wide range of cultured mammalian cells.
Interestingly, MGLs are not toxic to cells and can be sustainably
produced via microbial fermentation. This concept would play
a part in research into gene applications, and to make a
considerable contribution to gene therapies for HIV infection,
cancer and hereditary disorders. (This illustration was made
available courtesy of AIST, Japan).

133
C.O. Onwosi et al. Process Biochemistry 100 (2021) 124–139

for improved evaluation of biometabolite production [169]. Therefore,
the economic attainability of MGL production pathways from renewable
biomass should be considered in TEA. The importance of equipment
sharing between two product lines can also be beneficial in price
reduction [170]. Volumetric productivity is another essential determi­
nant of the economical sustainability of MGL production [21]. Accord­
ing to Zhang et al. [150] and Wang et al. [171], the evaluation of
fermentation performance using titer values may not be the most suit­
able approach. This is because the final titer tends to increase with the
longer fermentation processes (e.g., fed-batch fermentation operating
with in-situ downstream techniques). Therefore, productivity should be
a core part of TEA of MGL production because it gives a better evaluation
of yield than titer values as currently practiced. Wang et al. [161] also
suggested that TEA of MGL should center on variables rather than
having fixed values on certain parameters such as yield and selling price.
7. Application of MGLs in biomedical and personal care
products
Due to their attractive physicochemical properties (e.g., antimicro­
bial and anti-adhesive function, tensioactivity, CMC, self-assembly,
stability under harsh environmental conditions, etc.), MGLs have
found wide applications in the cosmetic industries and biomedicine.
Some of these interesting applications are summarized in Table 4. In this
section, we highlighted the mechanisms of action of MGLs in some
biomedical and personal care products.
7.1. Biomedical application
7.1.1. Potential therapy for pathological scar formation
Myofibroblast is a unique contractile fibroblast that has a significant
impact in the formation of pathological skin scar during wound healing
[191]. In the course of wound repair, myofibroblast performs two
essential functions – the expression of contractile mechanism such as α
-smooth muscle actin and secretion of collagen propolypeptides [191].
Expectedly, myofibroblast disappears through programmed cell death
after wound healing. However, in an unusual process of scarring, the
proliferation of myofibrolast continues and this leads to remodeling of
collagen fibre. In this situation, the aggregation and contraction of the
collagen fibres result in elevation of skin surface which eventually
makes the scar to overgrow. Not only does this hypertrophic scar have a
psychosocial impact among patients, but it also causes severe pain.
Interestingly, di-RL which has anti-fibrotic effects by collagen suppres­
sion that selectively kills the myofibroblast without damaging the
fibroblast (Fig. 3). This discovery corrected the earlier impression that
RLs inhibits the proliferation of dermal fibroblasts [191]. The discovered
mechanism of RL against scar formation would aid in the development
of innovative and functional pharmaceutical formulation towards

Fig. 5. (a) Moisturizing and (b) smoothening effects of MGL on cultured human skin model assay.
The effectiveness of MGLs in the recovery of damaged skin is comparable to natural ceramides. The use of the three-dimensional (3-D) cultured skin surface
accelerated the process for confirmation of the moisturizing properties of the MGLS. (The illustration was provided courtesy of AIST, Japan).

134
C.O. Onwosi et al.
fibrosis (e.g. scarring) remedy.
7.1.2. Increase in the efficiency of gene transfer into cells
Nano vectors (liposomes) are non-viral vectors useful in gene therapy
[192]. They are important tools for the delivery of foreign DNAs, small
interfering double-stranded RNAs, oligonucleotides and protein (anti­
bodies) into target cells (usually mammalian cells) with gene regulation
and transfection [193]. In the majority of experiments dealing with nano
vectors, cationic liposomes are most suitable owing to their low toxicity
and increased transfection efficiency [193]. Lipoplexes (lipo­
somes + DNA) – known to be unstable, heterogeneous and large – are
formed as a result of electrostatic interaction between negatively
charged pDNA and cationic liposomes [194]. Interestingly, the stability
as well as the performance of these agents (liposomes or lipoplexes) has
been reportedly improved by incorporating microbial-derived surfa­
ce-active agents [192,193,195]. As shown in Fig. 4,
liposomes-containing biosurfactants can be effective in the gene transfer
into different cultured mammalian cells [196]. MGLs are non-toxic to
the cells and can be produced from sustainable substrates. It is a very
vital approach currently utilized in biomedical research to treat different
ailments such as HIV infections, cancer and hereditary disorders [196].
When MEL congeners (MEL-A, MEL-B and MEL-C)-containing liposomes
were compared with regards to their effectiveness in membrane fusion
and DNA encapsulation, Ueno et al. [192] noted that MEL-A improved
both processes while MEL-B and MEL-C enhanced either of the two
processes. The estimated average diameters of liposomes such as 3 ([N-
(N′ , N′ –dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) or
cholesteryl-3β-carboxyamidoethylene-N-hydroxyethylamine (OH-Chol)
were 400–500 nm while MEL-A-containing nano vectors were
200–300 nm [197]. Inoh et al. [197] suggested that biosurfactants
improve transfection efficiency of the liposomes by reducing their par­
ticle size and the reduction of DNA-stimulated aggregation. Maitani
et al. [194] prepared injectable small-sized (300–350 nm) lipoplexes by
adding biosurfactants- MEL-A or β-sitosterol β-D-glucoside (Sit-G) (dis­
persants) to L-dioleoylphosphatidylethanolamine (DOPE)/ (DC-Chol)
liposomes. They noted that smaller-sized could be further obtained by
varying the sugar moiety or residue of MEL-A or Sit-G in the presence of
serum. They demonstrated that MEL and Sit-G-liposomes improved the
transfection efficiency of thymidine kinase and luciferase marker gene in
the presence of serum in the cell. Nakanishi et al. [195] developed lip­
oplex made up of MEL-A alongside DOPE and (OH-Chol) (cationic
cholesterol derivative). They demonstrated a novel pathway whereby
the biosurfactants facilitates gene delivery into targeted cells mediated
by the fusion of lipoplexes to the plasma membranes.
7.2. Personal care
7.2.1. MGLs as skin moisturizing agents
Most damage on the outermost skin layer (stratum corneum) leads to
a shortfall in ceramide content and the resultant loss of water retention
capacity. Topical treatments directed towards the reversal of the defect
usually utilize a large amount of natural ceramides which are expensive
[198]. To alleviate this concern, a skincare formulation with compara­
tively cheap ceramide-like active ingredients should be given attention.
Interestingly, the National Institute of Advanced Industrial Science and
Technology (AIST) Japan, together with Toyobo Cosmetics, developed a
three-dimensional cultured skin surface which helped to confirm that
MEL derivatives (MEL-A, -B and -C) have moisturizing effects on
damaged skin cells which were comparable to natural ceramide (Fig. 5a
and b) [199]. MELs, according to Bae et al. [200], also have restorative
action on ultraviolet (UV)-irradiation challenged cultured human
epidermal keratinocytes. This is due to its ability to modulate the action
of aquaporin-3 (AQP3) – a vital contributor to the moisturization of the
epidermis – which is downregulated in a UV-damaged skin.
135
Process Biochemistry 100 (2021) 124–139
8. Future prospects
Production of MGLs at yields matching those of industrially syn­
thesized surfactants (e.g. volumetric productivity of <2 gL− 1 h− 1) has
remained a major challenge for researchers over the past decade. While
major strides are currently being made by researchers, focusing more
research on improving the quality of the products, rather than their
quantity could be a more attractive perspective.
To expand the application range of MGLs and make them more
competitive against industrial surfactants, their structural variability
needs to be further elucidated. New techniques should be developed to
aid in the easy separation and identification of the congeners making up
the MGLs mix. These individual congeners should be analyzed to
delineate the main bioactive components inherent in the MGLs.
Furthermore, the possibility of manipulating the biosynthetic process for
increased ratios of the bioactive components in the MGLs mix should be
explored. While this may be a painstaking and time-consuming process
for researchers, it could lead to major strides in the production of tailor-
made biosurfactants for unique applications.
Advanced tools in genomics, transcriptomics, proteomics, and
metabolomics are indispensable in expounding the complete biosyn­
thetic pathways and their regulations in biosurfactant producing strains.
For example, altering the metabolic and biochemical processes of pro­
ducing organisms, usually by blocking the synthesis pathways of less
important metabolites thereby diverting the bulk of the energy towards
biosynthetic pathways of desired MGLs, could be an interesting
approach. Also, a more holistic genetic approach should be embarked
upon by researchers while bioprospecting for novel biosurfactant pro­
ducers. Unique gene clusters and fitness islands inherent in traditional
MGL producers should be identified and screening carried out on
industrially robust microorganisms to identify similar gene sequences.
As the biosafety concerns grow among consumers, novel non-pathogenic
microbial strains must be explored and utilized for large scale RLs and
other MGLs manufacturing [85]. Considering the microbial diversity
and dynamics in the environment, the current culturable organisms
constitute a very small fraction. In the light of this, pro-health organisms
could form the future biosurfactant market in biomedicine and personal
care [85].
While process and statistical optimization remain a successfully
implemented approach by researchers, the use of multiple enhancement
strategies during single production processes stands as a relatively new
perspective that could lead to new levels of structural variability and
unprecedented yields of biosurfactants. This will require developing
statistical models to simultaneously implement multiple improvement
strategies and identify the best combinations for specific MGLs types.
Due to the current advocacy for environmentally friendly paths for
the production of biometabolites, TEA should include LCA and resource
assessment during modeling of MGL production. To simplify the com­
parison of these pararmeters, the system boundaries should be uniform.
TEA should adapt equipment sharing and integrated processes for the
reduction in cost during evaluation of MGL production. As suggested by
Wang et al. [161], TEA of MGL should center on variables rather than
having fixed values on certain parameters such as yield and selling price.
9. Concluding remarks
MGLs hold enormous promises in cosmetics and biomedicine due to
their unique properties (e.g., antimicrobial and anti-adhesive attributes,
tensioactivity, self-assembly, stability over harsh environmental condi­
tions, etc.) and eco-compatibility. Despite growing interests in MGLs as a
reliable replacement for synthetic surfactants, production at commercial
scale has been hindered by high cost resulting from difficult downstream
processing or purification, complex regulation, biosafety concern,
structural heterogeneity and expensive feedstocks. To reduce cost and
enhance volumetric productivity, the MGL production process should be
more flexible. At the lab-scale, these adaptable bioprocceses have led to
C.O. Onwosi et al.
discovering novel MGL producers, developing platform organisms with
increased robustness for producing tailor-made MGLs, utilization of low-
cost feedstocks and in situ integrated product recovery. However,
replicating the results from laboratory-based investigations at com­
mercial scale is a complicated process and requires a decision-making
model (e.g., techno-economic assessment) to evaluate the economic
viability.
CRediT authorship contribution statement
Chukwudi O. Onwosi: Conceptualization, Project administration,
Resources, Supervision, Writing - original draft, Writing - review &
editing. Godwin O. Aliyu: Investigation, Methodology, Writing - orig­
inal draft. Chisom J. Onu: Investigation, Writing - original draft.
Kenechi O. Chukwu: Writing - original draft. Johnson K. Ndukwe:
Writing - original draft. Victor C. Igbokwe: Investigation, Writing -
original draft.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgement
The authors are grateful to AIST, Japan, for kindly providing dia­
grams used as Figs. 4 and 5 at no financial cost.
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