mannosylerythritol lipids bioproduction by · agradecimentos foi uma longa viagem, uma aventura...
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Mannosylerythritol lipids bioproduction by
Moesziomyces spp.: assessing alternative culture
strategies and nanofiltration downstream purification
Miguel Figueiredo Nascimento
Thesis to obtain the Master of Science Degree in
Biotechnology
Supervisors:
Prof. Dr. Frederico Castelo Alves Ferreira;
Dr. Nuno Ricardo Torres Faria
Examination Committee:
Chairperson: Prof. Drª Helena Maria Rodrigues Vasconcelos Pinheiro
Supervisor: Dr. Nuno Ricardo Torres Faria
Member of the committee: Drª Susana Santos Moita de Oliveira Marques
November 2017
Agradecimentos
Foi uma longa viagem, uma aventura incrível e, em primeiro lugar, gostaria de agradecer aos
meus supervisores, Professor Drº Frederico Ferreira, por me introduzir um tema tão interessante
e desafiante como o “MEL”e me dar a oportunidade de trabalhar nos laboratórios do IBB, bem
como pelas ideias incriveis criadas ao longo deste ano e pelo continuo apoio; ao Drº. Nuno Faria,
por me guiar em cada experiência no laboratório, pela motivação, pelas perguntas criadas a cada
ideia minha e por nunca me deixar ir abaixo, mostrando-me sempre o caminho correcto, e,
obviamente aos grandes jogos de futebol disputado durantes as várias semanas deste ano. Um
grande obrigado aos dois por tudo.
Em segundo lugar, gostava de agradecer ao Flávio Ferreira, pelo seu conhecimento, por me
ensinar todas os mecanismos associados a nanofiltração e acima de tudo pelas várias horas que
dispensou para me ajudar. De seguida, gostava tambem de agradecer a Marisa Santos e
Margarida Silva por me ensinarem todas as técnicas relacionadas com a produção de “MEL”, a
paciência e o entusiamo sempre disponivel. Ao António Maduro, Dona Rosa, Ricardo Pereira,
Drª Carla Carvalho e Drº Pedro Fernandes por toda a assistência.
Gostava de agradecer ao financiamente pela Fundação para a ciência e tecnologia (FCT)
através do projeto Cruise: Pseudozyma spp based refinery: Membrane bioreactor for production
of aviation fuel and biosurfactants, PTDC/AAG-TEC/0696/2014; IBB- Instituto de bioengenharia
e ciências (Referência FCT: UID/BIO/04565/2013 UID/BIO/04565/2013 e POL2020, referência
007317, incluindo iBB ITACYEAST) e á bolsa de estudos SFRH/BPD/108560/2015, permitindo-
me obter todos os recursos necessários para desenvolver esta tese de mestrado.
De seguida gostava de agradecer aqueles que nunca falhar, especialmente aos “Abadia”, aos
meus três melhores amigos – Nuno Marques, Carlos Fernandes e Miguel Chapado -, pelas
grandes conversas, caminhadas, e especialmente, por estarem presente na minha vida.
Obviamente que não podia deixar de agradecer aos “gunas”, sem vocês estes dois anos não
teriam sido tão divertidos e entusiasmantes! Um grande obrigado por terem estado sempre
presentes.
À Mariana São Pedro, madrinha de praxe, por me ajudar desde o primeiro ano de faculdade.
Sei que no início era só eu e tu, mas a familia “aumentou” e agora somos seis elementos, um
grupo de pessoas loucas e felizes. Não podia estar mais orgulhoso por ter a oportunidade de vos
conhecer a todos, um grande obrigado “Muchachily”.
Ao Tiago Magalhães, pelo apoio e pelas conversas longas sobre biotecnologia e psicanálise
e, acima de tudo, por estar sempre presente para ouvir todos os meus problemas.
Um obrigado gigante à minha equipa de futebol, “CAO-Clube Académico de Odivelas”, aos
meus companheiros de equipa por me ajudarem a esquecer todos os problemas durantes os
treinos e jogos.
À Moradia, a aldeia mais espetacular, aos meus avós por serem o meu orgulho e me darem
a força necessaria para continuar a percorrer este caminho. Aos meus primos, com especial
referência ao Rui Nascimento pelos verões passados juntos.
ii
Finalmente, às pessoas mais importantes na minha vida:
À Inês Cachola: obrigado por seres única, por tornares a minha vida melhor, mais colorida,
mais alegre, por seres a minha companheira de todos os momentos, por todas as nossas viagens
pela Europa, por me “aturares”, suportares, e, sobretudo, amares.
Aos meus pais, Ernesto Nascimento e Cristina Nascimento por tudo: por serem das pessoas
mais inteligentes que já conheci, por me ensinarem tudo e me terem ajudado a chegar onde
cheguei. E à minha irmã, Leonor Nascimento, por conseguires animar-me sempre e,
principalmente, por seres quem és. Sei que nem sempre tenho o melhor humor, sei que nem
sempre sou a melhor pessoa, mas representam tudo para mim e não há palavras que descrevam
o quanto me deixam feliz. Obrigado.
iii
Abstract
The aim of this work consisted in studying alternative approaches for production of
mannosylerythritol lipids (MEL), using Moesziomyces antarcticus and Moesziomyces aphidis. To
achieve this aim, different carbon sources to increase MEL production and decrease the final
concentration of fatty acids (FA) were used. Also, nano-membranes were used to separate both
components.
This thesis has demonstrated, from the conditions tested, that the highest yields are reached
when the fermentation process begins with 40 g/l of glucose and, after four days of bioconversion,
40 g/l of soybean oil is added, where it was obtained a MEL titre of 24.7±2.5 g/l for M. antarcticus
and 17.6±1.6 g/l for M. aphidis, with a yield of 0.31±0.03 and 0.22±0.02 gMEL/gsubstrate, respectively,
after 14 days in a shake flask.
Production of MEL, using waste frying oils (WFO), resulted on MEL titre of 10.0±0.1 g/l for M.
aphidis and 12.1±0.5 g/l for M. antarcticus, with a yield of 0.17±0.00 and 0.20±0.01 gMEL/gsubstrate,
after 14 days in a shake flask.
The production of MEL by M. aphidis was 12.58 g/l after 12 days of bioconversion in a
bioreactor, with a yield of 0.20 gMEL/gsubstrate. With M.antarcticus, at day 5, a titre of 10.54 g/l of
MEL was obtained, corresponding to a maximum productivity of 0.09 g/l/h. After this day, the
substrate was consumed, and MEL production decreased.
Considering downstream, a nanofiltration membrane (540-580 Da) was assessed to MEL/FA
separation, obtaining a rejection coefficient of 98% for MEL and 60% for monoglycerides.
KEYWORDS: Biosurfactants; Mannosylerythritol Lipids; Bioreactors; Nanofiltration
technology;
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v
Resumo
O objetivo deste trabalho consistiu em estudar abordagens alternativas para a produção de
manosileritritolípidos (MEL), usando Moesziomyces antarticus e Moesziomyces aphidis. Tendo
em conta o objetivo, usou-se diferentes fontes de carbono para aumentar a produção de MEL,
diminuindo a concentração final de ácidos gordos no meio de cultura. Usou-se nano-membrans
para separar MEL dos ácidos gordos.
Demonstrou-se que de todas as condições testadas, a melhor foi aquela em que se inicia a
fermentação com 40 g/l de glucose e, ao final do quarto dia, o meio é suplementado com 40 g/l
de óleo de soja, obtendo-se uma concentração de 24.7±2.5 g/l de MEL para a M. antarcticus e
uma concentração de MEL de 17.6±1.6 g/l para a M. aphidis, com um rendimento de 0.31±0.03
e 0.22±0.02 gMEL/gsubstrato, respetivamente, depois de catorze dias em frascos agitados.
Recorrendo-se ao uso de óleos de fritura usados, obtêm-se uma concentração de MEL de
10.0±0.1 g/l para M. aphidis e 12.1±0.5 g/l para a M. antarcticus, com um rendimento de
0.17±0.00 e 0.20±0.01 gMEL/gsubstrato, respetivamente, depois de catorze dias em frascos agitados.
Em bioreactores, recorrendo a M. aphidis, obtêm-se uma concentração de 12.58 g/l de MEL.
Com M. antarticus, ao dia cinco, obteve-se uma concentração de 10.54 g/l de MEL,
correspondendo ao máximo de produtividade (0.09 g/l/h).
No processo de recuperação, usou-se membrana de nanofiltração (540-580 Da) para permitir
a separação de MEL/FA. Obteve-se um coeficiente de rejeição de 98% para o MEL e 60% para
os monoglicerídeos.
PALAVRAS CHAVE: biosurfactantes; manosileritritolípidos; Biorreatores; Tecnologia de
Nanofiltração;
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vii
Table of contents
Agradecimentos ........................................................................................................ i
Abstract .................................................................................................................... iii
Resumo .................................................................................................................... v
List of tables ............................................................................................................ ix
List of figures ........................................................................................................... xi
List of abbreviations ............................................................................................... xv
Chapter 1 - Introduction ............................................................................................ 1
1.1 Overview ......................................................................................................... 1
1.2 Objectives and challenges .............................................................................. 1
1.3 Research questions and research strategies: ................................................. 2
Chapter 2 - Literature review and State-of-the-art .................................................... 4
2.1 Surfactants and their applications in the global market ....................................... 4
2.1.1 Surfactants................................................................................................... 4
2.1.2 Market assessment ...................................................................................... 4
2.2 Biosurfactants and their applications in the market ............................................. 6
2.2.1 Biosurfactants .............................................................................................. 6
2.2.2 Market assessment of microbial biosurfactants ............................................ 9
2.2.3 Renewable substrates and their use to produce biosurfactants.................. 11
2.3 Mannosylerythritol Lipids (MEL) ....................................................................... 12
2.3.1 Why MEL? ................................................................................................. 12
2.3.2 Metabolic pathways for producing MEL ...................................................... 13
2.3.3 Applications of MEL ................................................................................... 14
2.4 Fermentation processes to produce MEL ......................................................... 15
2.4.1 Influence of carbon source in the production of MEL .................................. 15
2.4.2 Influence of nitrogen source in the production of MEL ................................ 16
2.4.3 Scale-up the production of MEL in bioreactors ........................................... 16
2.5 Downstream processing of biosurfactants ..................................................... 17
2.5.1 Downstream processing of MEL ............................................................. 18
Chapter 3 - Material and Methods .......................................................................... 20
3.1 Materials ....................................................................................................... 20
3.2 Microorganisms and maintenance ................................................................ 20
3.3 Media and Cultivation conditions................................................................... 20
3.4 Shake flask cultivation .................................................................................. 20
3.5 Bioreactor cultivation ..................................................................................... 21
3.6 Lipolytic assay .............................................................................................. 22
3.6.1 Enzymatic reaction using Lipase B (CAL-B) ........................................... 22
3.7 Cell growth .................................................................................................... 22
3.8 Sugar profile ................................................................................................. 23
3.9 Quantification of MEL .................................................................................... 23
3.9.1 Methanolysis and GC analysis ............................................................... 23
3.9.2 MEL extraction ....................................................................................... 23
3.9.3 TLC analysis .......................................................................................... 23
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3.10 Nanofiltration ............................................................................................... 24
3.10.1 Glycerides quantification ...................................................................... 24
Chapter 4 - Results and discussion ........................................................................ 25
4.1 Studying the effect of using two carbon sources in the production of MEL .... 25
4.1.1 MEL production using SBO .................................................................... 25
4.1.2 Pulses of two carbon sources (hydrophilic and hydrophobic) to increase
MEL titres. ........................................................................................................... 28
4.1.3 Development of a fed-batch strategy for M. aphidis and M. antarcticus
cultivation using hydrophilic and hydrophobic carbon source ............................... 32
4.1.4 Producing MEL using compounds enrichment with nitrogen ................... 35
4.1.5 Lipolytic activity ...................................................................................... 39
4.2 Production of MEL by mixed carbon source strategy utilization in bioreactors
................................................................................................................................ 41
4.2.1 Lipolytic activity in bioreactors ................................................................ 46
4.3 Producing MEL using waste frying oil (WFO) ................................................ 47
4.4 Downstream processing by nanofiltration technology .................................... 49
4.4.1 Testing the membrane with 22% of PBI solution..................................... 49
4.4.2 Enzymatic reaction to breakdown triglycerides ....................................... 52
4.4.3 Testing the membrane with 17% of PBI .................................................. 54
Chapter 5 - Conclusions ......................................................................................... 57
Chapter 6 - Future perspectives ............................................................................. 59
Chapter 7 - Bibliography ......................................................................................... 61
Chapter 8 - Appendix ............................................................................................. 70
8.1 Appendix 1 .................................................................................................... 70
8.2 Appendix 2 .................................................................................................... 71
8.3 Appendix 3 .................................................................................................... 72
8.4 Appendix 4 .................................................................................................... 73
8.5 Appendix 5 .................................................................................................... 74
ix
List of tables
Table 1: Methods for screening of biosurfactants-producing microorganisms (adapted from22):
....................................................................................................................................................... 7
Table 2: Resume of the 5 classes of biosurfactants, including some examples of each class,
and the respectively microorganism producer. Table adapted from5 ............................................ 8
Table 3: Some of the largest microbial biosurfactant producing companies around the world.
Adapted from44 .............................................................................................................................. 9
Table 4: Summary of the several renewable substrates from industry with the potential to be
used as a carbon source. Adapted from6 .................................................................................... 11
Table 5: Summary of maximum MEL obtained, yield (product/substrate), yield in mol, purity
factor and productivity for each condition with M. aphidis and M. antarcticus. ........................... 27
Table 6: MEL and FA titres, yields, purity factor and productivities in 14 days fed-batch
cultivation of M. antarcticus and M. aphidis. The condition marked at bold, represents the
extraction of MEL and FA from the all broth. ............................................................................... 32
Table 7: Summary of maximum MEL obtained, yield (product/substrate), yield in mol, purity
factor and productivity for each condition in M. aphidis and M. antarcticus. ............................... 35
Table 8: Summary of MEL obtained at day 14th of fermentation, yield (product/substrate), yield
in mol, purity factor and productivity for peptone, CSL yeast extract and the control ([0:40sbo] with
the normal components) for M. aphidis and M. antarcticus. ....................................................... 38
Table 9: Summary of MEL obtained at day 14th of fermentation, yield (product/substrate), yield
in mol, purity factor and productivity for conditions 1 feed of 20g/l of SBO in bioreactor and in
shake flask, and for the condition with feeds of 2g/l.................................................................... 43
Table 10: Resuming of the MEL obtained at day 10th of fermentation for shake flask, and day
5th of fermentation for the bioreactor. Also, the yield (product/substrate), yield in mol, and
productivity. ................................................................................................................................. 45
Table 11: MEL and FA production, yields, purity factor and productivity after 14 days of M.
aphidis and M. antarcticus cultured on 40 g/l of glucose and pulse of WFO at day 4. ............... 48
Table 12: Theoretical calculation of concentration in retentate (cR) for FA and MEL, % of FA
in the feed and MEL purity (%), assuming a rejection coefficient for MEL and FA of 98% and 60%,
respectively and a concentration of MEL (14.85 g/l) and FA (3.85 g/l) ....................................... 54
x
xi
List of figures
Figure 1: World consumption of surfactants in 2015, retrieved from 19. ................................... 5
Figure 2: Major consumption of surfactants by major application area. Retrieved from1 ........ 5
Figure 3: Compilation of the largest biosurfactant producer companies around the world, in
each country. Adapted from41 ...................................................................................................... 10
Figure 4: Biosurfactants market volume share, by application in 201324. .............................. 10
Figure 5: Chemical structure of MEL and their types. MEL-A : di-acylated; MEL-B: mono-
acylated in C6; MEL-C: mono-acylated in C456 ........................................................................... 12
Figure 6: Resume of possible metabolic pathways to produce MEL. Retrieved from66 ......... 14
Figure 7: Images of culture medium for the condition 60 g/l of SBO in M. antarcticus at
14th day of fermentation 26
Figure 8: Result obtained by TLC: a) Comparison of MEL extracted from M. antarticus and M.
aphidis; b) Comparison between MEL extracted from M. aphidis and aggregate from fermentation
broth. ........................................................................................................................................... 26
Figure 9: Maximum production of MEL (White bars), fatty acids (black bars) and cell dry weight
(grey bars) in each condition (80, 60, 40 and 20g/l of SBO) for M. aphidis. ............................... 27
Figure 10: Maximum production of MEL (White bars), fatty acids (black bars) and biomass
(grey bars) in each condition (80, 60, 40 and 20g/l of SBO) for M. antarcticus .......................... 27
Figure 11: Production of MEL (a), consumption of FA (b), formation of biomass (c) by M.
aphidis; Production of MEL (d), consumption of FA (e), formation of biomass (f) by M. antarcticus
for the conditions: [40glu,0:40glu4] (Dashed line with ); [40glu,0:40glu and 5sbo,4] (Line
with▲); [40glu,0:40glu and 10sbo,4] (Dashed line with ●) and [40glu,0:40glu and 20sbo,4] (Line
with ■). Red markers means the presence of red aggregates in culture medium. ..................... 29
Figure 12: Evolution of the red aggregates in M. aphidis cultivation for the condition
[40glu,0:40glu and 20sbo,4] at: a) day 7; b) day 10; c) day 14 of fermentation .......................... 30
Figure 13: Typical behaviour for glucose consumption for M. aphidis (grey line) and
M.antarcticus (black line) ............................................................................................................. 31
Figure 14: Production of MEL (a), consumption of FA (b), formation of biomass (c) and glucose
consumption (d), by M. aphidis; Production of MEL (e), consumption of FA (f), formation of
biomass (g) and glucose consumption (h), by M. antarcticus for the conditions: [40sbo,0:40glu,4]
(Dashed line with ▲); [40glu,0:20sbo,4] (Line with ●) and [40glu,0:40sbo,4] (Line with ■). Red
markers represent the presence of red aggregates in cultivation. .............................................. 33
Figure 15: Production of MEL (a), consumption of FA (b), formation of biomass (c) and glucose
consumption (d) by M. aphidis; Production of MEL (e), consumption of FA (f), formation of
biomass (g) and glucose consumption (h) by M. antarcticus for the conditions: peptone (Dashed
line▲); yeast extract (Dashed line with ●) and corn steep liquor (Line with ■). .......................... 37
Figure 16: Extracellular lipolytic activity profile determined in M. aphidis cultured for the
conditions: a) [80g/l SBO] (Dashed line with ▲) and [40glu,0:40glu,4] (Line with ●); b)
[40glu,0:40sbo,4] (Line with ) and [40sbo,0:40glu,4] (Line with ▲). ........................................ 39
xii
Figure 17: Extracellular lipolytic activity profile determined in M. antarcticus cultured in the
conditions: a) [80g/l SBO] (Dashed line with ▲) and [40glu,0:40glu,4] (Line with ●); b)
[40glu,0:40sbo,4] (Line with ) and [40sbo,0:40glu,4] (Line with ▲). ........................................ 40
Figure 18: Production of MEL in bioreactors with M. aphidis, starting with 40g/l of glucose and
1 feed of 20g/l of SBO after the first day. a) MEL production (Line with ●) and FA consumption
(Dashed line with ▲); b) Glucose consumptions (Dashed line with ▲) and biomass (Grey Line
■). Red figures represent the days that appeared aggregates. .................................................. 41
Figure 19: Evolution of the red aggregates in M. aphidis cultivation in bioreactor using 40 g/l
of glucose and after 1 day, 20 g/l of soybean oil were fed: a) day 4; b) day 5; c) day 6 and d) day
9 of fermentation.......................................................................................................................... 42
Figure 20: Production of MEL in bioreactor with M. aphidis, adding 2g/l of SBO for 10 days: a)
MEL production (Blue line ●) and FA consumption (Black line ▲); b) Glucose consumptions
(Dashed line with ▲) and biomass (Grey line ■). ....................................................................... 42
Figure 21: Production of MEL in bioreactor with M. antarcticus, adding one feed of 20g/l of
SBO: a) MEL production (Blue line ■) and FA consumption (Black line▲); b) Glucose
consumptions (Dashed line with ●) and biomass (Grey line ■). ................................................. 44
Figure 22: Production of MEL in bioreactor with M. antarcticus, adding one feed of 20g/l of
SBO: a) MEL production (Blue line ■) and FA consumption (Black line▲); b) Glucose
consumptions (Dashed line with ●) and biomass (Grey Line■). ................................................. 44
Figure 23: Image of the biofilm formed in bioreactor with M. antarcticus .............................. 45
Figure 24: Extracellular lipolytic activity profile determined in M. aphidis cultured on 40 g/l of
glucose and: 1 pulse feed of 20 g/l of SBO (Black line ■) and several pulse feeds of 2 g/l (Dashed
line with ●). .................................................................................................................................. 46
Figure 25: Extracelular lipolytic activity profile determined in M. antarcticus cultured on 40 g/l
of glucose and 1 pulse feed of 20 g/l of. ..................................................................................... 47
Figure 26: Production of MEL (Dashed line with ▲), consumption of FA (Line with ▲),
formation of biomass (■) and glucose consumption (Dashed line with ●) by M. aphidis for
conditions [40glu,0:20wfo,4]. ....................................................................................................... 47
Figure 27: Production of MEL (Dashed line with ▲), consumption of FA (Dashed line with ▲),
formation of biomass (Line with ■) and glucose consumption (Dashed line with ●) by M.
antarcticus for conditions [40glu,0:20wfo,4]. ............................................................................... 48
Figure 28: Flux for each condition: 10 bar ethyl acetate (black bar), 20 bar ethyl acetate (blue
bar), 30 bar Ethyl acetate (grey bar); MTBE (orange bar) and isopropanol (green bar) ............. 50
Figure 29: Rejection coefficient of MEL for each condition: 10 bar ethyl acetate (black bar), 20
bar ethyl acetate (blue bar), 30 bar Ethyl acetate (grey bar); MTBE (orange bar) and isopropanol
(green bar) ................................................................................................................................... 50
Figure 30: Rejection coefficient for monoglycerides (a) and triglycerides (b) for each condition:
10 bar ethyl acetate (black bar), 20 bar ethyl acetate (blue bar), 30 bar Ethyl acetate (grey bar);
MTBE (orange bar) and isopropanol (green bar) ........................................................................ 51
xiii
Figure 31: Percentage of masses and losses for MEL (a) and FA (b), for the conditions 10 bar
ethyl acetate (black bar), 20 bar ethyl acetate (blue bar), 30 bar Ethyl acetate (grey bar); MTBE
(orange bar) and isopropanol (green bar). Orange bars represent the losses of the compounds.
..................................................................................................................................................... 51
Figure 32: HPLC spectre of the aggregates of MEL and FA from the bioreactor with M.aphidis
and used to perform filtrations. Blue rectangle correspond to MAG and red rectangle corresponds
to TAG. ........................................................................................................................................ 52
Figure 33: HPLC spectre, after the enzymatic reaction have occurred. Blue rectangle
corresponds to MAG, and red rectangle corresponds to the zone, where TAG should appear. 53
Figure 34: Flux for each condition: organic phase (black bar) and aqueous phase (blue bar)
..................................................................................................................................................... 54
Figure 35: Rejection of MEL for both phases. Organic phase (black bar) and aqueous phase
(blue bar) ..................................................................................................................................... 55
Figure 36: Rejection coefficient for organic phase and aqueous phase: a) monoglycerides and
b) triglycerides ............................................................................................................................. 55
xiv
xv
List of abbreviations
Acetyl-CoA – Acetyl Coenzyme A
Ca2+– Calcium ion
CAC – Critical aggregates concentration
CAGR – Compound annual growth rate
CDW – Cell dry-weight
CF – Concentration in feed
CMC – Critical micelle concentration
cR – Concentration in retentate
CSL – Corn steep liquor
D – Diavolumes
DAG - Diglycerides
FA – Fatty acids
GC – Gas Chromatography
GDP-mannose – Guanosine diphosphate mannose
Li+– Lithium ion
MAG – Monoglycerides
MEL – Mannosylerythritol lipid
MTBE – tert-butyl methyl ether
MWCO – Molecular weight cut-off
Rc – Rejection coefficient
SBO – Soybean oil
TAG – Triglycerides
TLC – Thin layer chromatography
TNF-α – Tumour necrosis factor
USD – United State dollars
WFO – Waste frying oil
WWTP – Wastewater treatment plant
xvi
1
Chapter 1 - Introduction
1.1 Overview
Since the industrial revolution, many industries have raised and increased through the years,
especially the chemical industry, extracting petroleum and producing several compounds to be
used in numerous applications.
Surfactants are one of the most produced and consumed chemicals all over the world. They
are compounds with unique characteristics and are used in a wide range of products, like
detergents, household products and motor oils. This is a market in expansion, where it is projected
to reach 39.86 USD billion in 20211,2. However, over the last decades, the increase on the
awareness of the importance of the need for a sustainable production and environment concerns
on the chemical impact have led the consumers behaviour and companies taking into account
decisions regarding the environment.
Considering the recent advances, biotechnology can provide solutions to those problems by
using wild microorganisms able to produce the same compound or engineering strains to produce
a given compound. In fact, there are many groups and companies, as well as public funds in
European Union investing in green technologies3.
Microbial biosurfactants are the most promising compounds to replace chemical surfactants
due to their lower environmental impact and high biodegradability4,5.
These advantages, coupled to the use of renewable substrates, rather than production from
petroleum, contribute to increase their sustainable production. Cost reductions can be envisaged
with a scale up of their industrial production and with the use of waste materials as substrates
(renewable substrates)6.
Nowadays, microbial biosurfactants being produced from renewable substrates include
sophorolipids, rhamnolipids and mannosylerythritol lipids (MEL). In fact, the first two microbial
biosurfactants are well established in the market, which combined market is projected to reach
USD 17.5 million by 2020, with an annual growth of 4%7.
Among the microbial biosurfactants produced and studied, MEL (the biological product
targeted in this project) is a promising product to reach the market due to the numerous of
applications in a variety of fields. However, there are some challenges associated with the
industrial production of MEL, such the downstream processing. So, it is necessary to develop a
new integrated bioprocess, improving the fermentation process and/or the downstream
processes, to decrease manufacturing costs.
1.2 Objectives and challenges
MEL has been usually produced using vegetable oils as carbon source, such as soybean oil
(SBO), which leads to higher product titres, but also to high amount of fatty acids in the end of
fermentation, which leads to major difficulties in the downstream processing8, 9.
2
To adress this problem, production of MEL using sugars (like glucose) and renewable
lignocellulosic residues offers a medium without fatty acids. However the titre of MEL reached in
the end of fermentation is considerably lower10,11.
The global aim of this thesis was to study and improve the fermentation process using two
yeast species, Moesziomyces aphidis and Moesziomyces antarcticus, observing the differences
of using two different carbon source addition in shake flask and bioreactors. In addition, it was
performed a separation method of MEL and fatty acids.
Basically, the present work (here reported), includes the study of:
• Fermentation process, improving and studying the fermentation process combining two
types of carbon source, hydrophilic (glucose) and hydrophobic (SBO or WFO). Aiming to
obtain high titres of MEL and reaching a lower concentration of fatty acids in the end of
the fermentation. Also, the effect of others sources was studied, such as corn steep liquor,
peptone and yeast extract. This investigation involves the study of MEL production in
shake flask and bioreactors.
• Downstream process: After the fermentation, MEL and FA were extracted with ethyl
acetate and the solvents are evaporated and these aggregates were solublized in
differents organic solvents. Considering that in the end of fermentation the fatty acids
existing in the medium are composed by monoglycerides, diglycerides and triglycerides
with differents sizes, a nanofiltration step was assessed to separate MEL from FA.
1.3 Research questions and research strategies:
This thesis addresses the following questions:
1. Can a pulse of two carbon sources (hydrophilic and hydrophobic) improve MEL titres?
2. Does the order of carbon source (hydrophilic or hydrophobic) affect MEL titre?
3. Increasing rich medium suppliers (such as corn steep liquor and peptone) can
stimulate the consumption of fatty acids and increase MEL titres?
4. Can nanofiltration (NF) be used to separate fatty acids from MEL?
5. Which specie should be used to produce MEL in bioreactor?
To answer the five research questions, the following experiments were performed:
• MEL is usually produced, using vegetable oils, namely SBO. Therefore, in a 1st set of
experiments different concentrations of SBO were tested (80, 60, 40 and 20 g/l) to
assess the effect of this carbon source in productivity of MEL and the level of
contamination of fatty acids in the end of fermentation.
• Faria et al11 and Morita et al12 have described the production of MEL using hydrophilic
compounds (glucose and xylose) instead of the usual vegetable oils. With this
approach, the fermentation ends with almost no fatty acids, even though, the yields of
MEL are lower (0,075 g/g). To understand if the combination of two carbon sources
(hydrophilic and hydrophobic) can increase MEL titres and finish the fermentation with
3
lower FA, a 2nd set of fermentation experiment started with 40 g/l of glucose and at the
4th day of fermentation, pulses of SBO (5, 10, and 20 g/l) and glucose (40 g/l) were
added to the medium.
• To assess if the order of addition of hydrophobic/hydrophilic compounds to the
fermentation affects MEL titre, a 3rd set of fermentation experiment were performed,
studying three conditions: Start with 40 g/l of glucose and supply with 40 g/l of SBO
at 4th day; start with 40 g/l of SBO and supply with 40 g/l of glucose at 4th day; and,
finally, start with 40 g/l of glucose and supply with 20g/l of SBO at 4th day.
• The importance of the ratio Carbon/Nitrogen to the production of MEL is well
described, as described by Faria et al 13 and Rau et al14. To assess if the addition of
rich compounds to the medium could stimulate MEL production and FA consumption,
peptone, yeast extract and corn steep liquor were added to the medium, with a
concentration of 10 g/l. For this assay, the fermentation started with 40 g/l of glucose
and 40 g/l of SBO were supplied at 4th day.
• Producing a given compound in a bioreactor is not necessarily equal to the production
observed in shake flasks. So, from the conditions tested in shake-flask, the one
capable of producing higher titres of MEL and maintaining a low concentration of FA
in the end of fermentation was tested in a bioreactor. It was also evaluated the
performance of both species in a bioreactor.
• Most of the studies have used vegetable oils to produce MEL, obtaining relatively high
titres of MEL8,9. Although, to separate MEL from FA it is not easy, and multiple solvent
extraction are necessary, as described by Rau et al9 (see sector 2.5.1), obtaining only
8% of pure MEL. So, in this way nanofiltration technology was tested to understand if
it is possible to separate MEL from FA.
4
Chapter 2 - Literature review and State-of-the-art 2.1 Surfactants and their applications in the global market
2.1.1 Surfactants
Nowadays our society lives in an industrialized world with a variety of industries, where
chemical industry is one of the largest manufacturing industries and surfactants are among the
chemicals produced15.
Surfactants (surface active agents) are molecules with a hydrophobic tail and a hydrophilic
head, known as amphipathic structure. These compounds adsorb in the interface or surface,
forming tightly packed structures 16. Mainly, when the solvent is water, the tendency of surfactants
is to minimize the contact between water and then hydrophobic group, starting a process called
“micellization”. This process involves the aggregation of surfactants (micelles), with their
hydrophilic group toward the aqueous solution, starting at very low concentration, which is known
as critical micelle concentration (CMC)1.
The term interfaces indicate a boundary between two immiscible phases, existing 5 types of
interface: solid-vapor surface, solid-liquid, solid-solid, liquid-vapor surface and liquid-liquid 17.
Therefore, these surfactants can reduce the surface and interface tension, increasing the
solubility of hydrophobic compounds in an aqueous media or the solubility of water in a
hydrophobic solution (hydrocarbons). According to the hydrophilic group of surfactants, these
compounds can be classified as anionic, cationic, zwitterionic and non-ionic1,17.
Due to these properties, surfactants have the ability of detergency, foaming, emulsification/de-
emulsification, dispersion/aggregation of solids, adsorption and micellization5. Consequently,
surfactants have a wide range of applications, being used in a variety of products such as
detergents, household products and motor oils. Considering the efficiency of surfactants in
removing dirtiness, the more representative chapter for application of this group of chemicals is
the formulation of detergents (see figure 2).
Regarding all the surfactants produced, there are a type of surfactants, “gemini” surfactants,
which have been gaining importance in the industry, since the 1980. This surfactant is a dimeric
surfactant, constituted by two hydrophilic groups and two tails18 and, due to this constitution, these
surfactants have a lower CMC than the rest of the surfactants produced. In others words, all the
properties mentioned earlier are better when compared with others surfactants16.
2.1.2 Market assessment
In the last few decades, the demand for surfactants increased about 300% and the surfactant
market in 2016 was evaluated in USD 30,84 billion and it is projected to reach 39,86 USD billion
in 2021 2.
Surfactants are consumed all over the world, with special relevance in North America, Europe
and Asia, as described in figure 1. Although, regarding the pressures and restricted rules in
Europe and United States of America, this market is changing, in Pacific Asia, where it is expected
to grow with a CAGR of 6,1% until 2020.
5
Figure 1: World consumption of surfactants in 2015, retrieved from19.
The formulation of detergent and household products is the application that consumes more
surfactants. Namely, 54% of the surfactants produced in the world are being used in household
products, as described in figure 2. Of the remaining surfactants produced, 41% are used in
industry, being mostly used as biocides and tank cleaners; 8% are used in personal care like
shampoos, cosmetic creams or perfumes.
Figure 2: Major consumption of surfactants by major application area. Retrieved from1
These surfactants derive from petroleum, and some of them have the ability to bind to
components of the cell, such as liposomes. Bragadin et al20 have shown that the surfactant linear
alkylbenzene sulfonate have the ability to accumulate in liposomes from rat liver. Although, the
effect of surfactants in the environment vary accordingly to the type of surfactants21. For example,
Alkylphenol ethoxylates (APE) belongs to the non-ionic class of surfactants and are one of the
most used, and after the primary treatment in wastewater tretatment plant (WWTP), leads to the
formation of alkylphenols (e.g. polyphenol, octylphenol)22. These metabolites have tendency to
bioaccumulate in the lipids of organism and there are reports of the accumulation in aquatic
species 22,23. Although, more studies are required to evaluate the toxicity of surfactant and the
best treatment to apply in WWTP and avoid the accumulation in air, soil or aquatic compartments.
6
Currently, there is a great concert regarding the sustainability and global warning. Therefore,
microbial biosurfactants, have gain attention due to their excellent properties, especially
biodegradability, and could replace common surfactants.
2.2 Biosurfactants and their applications in the market
2.2.1 Biosurfactants
Most of the biosurfactants are surfactants obtained by chemically reaction from vegetable
products24 and among these, there are a sub class of biosurfactants, microbial biosurfactants,
which are surface active compounds synthesized by microorganisms. The microbial production
of these compounds, plays, in some cases, an important role in the defence and protection of the
cell, by disrupting the cell membrane of others microorganism25, increasing the surface area of
the microorganism by facilitating nutrient uptake and biofilm formation or promoting motility of the
microorganisms (e.g surfactin)26,27.
Microbial biosurfactants, like fossil driven surfactants, have hydrophobic and hydrophilic
regions allowing them to reduce surface and interfacial tension by the same mechanism of
surfactants already used industrially. Depending on the composition of these compounds, they
can be used as an effective emulsifier, for detergency, like described earlier for surfactants4,5.
The demand for green products have increased and the final consumers are getting conscious
about the problems of using fossil driven chemicals to the environment and human health.
Therefore, biosurfactants are alternatives to replace these chemical surfactants, mainly due to
their biodegradability (ability to be synthetized using renewable substrates), presenting less
damage to the environment, but also due to other important properties such as a better tolerance
to high temperatures, pH and salinity; as well the ability of foaming, which allows to expand the
application scope4,5.
With the recent advances in the fields of genomics and metagenomics, the number of methods
for screening biosurfactant-producing microorganisms have increased, as described in table 1.
Consequently, the number of biosurfactants have increased through the years and it was
necessary to categorize these compounds, accordingly to their chemical and physical properties.
7
Table 1: Methods for screening of biosurfactants-producing microorganisms (adapted from22):
Reference Analytical method Description of the method
Cooper and Gold-
enberg28 Emulsification test
Estimation of the emulsification value
(E-24), only valid for emulsifying
biosurfactants
Matsuyama et al29 Droplet on slides Method that uses TLC (thin-layer
chromatography)
Shulga et al30 Colorimetric methods
Colorimetric method based on the
ability of anionic surfactants to react
with the cationic indicator, forming a
complex.
Lindhal et al31 Salt aggregation test
Precipitation of cells with salts. The
order in which cells are precipitated is
a measure of their surface
hydrophobicities
Rotenberg et al32 Bacterial adhesion to
hydrocarbon compounds
This method is based on the degree
of adherence of cells to various liquid
hydrocarbons
Vaux and
Cottingham33 Microplate method
A light beam is passed through the
sample in the microplate. Surface
tension is measured by quantifying
the intensity of light reflected
Jain et al34 Drop collapsing test
Sensitive and rapid method to screen
for bacterial colonies producing
surfactant. Solutions containing
potent surfactants will be unable to
form stable drops on an oily surface
Van der Vegt et al35 Axisymmetric drop shape analysis by profile
Technique that determines the
contact angle and liquid surface from
a droplet resting on a solid surface
There are five major classes of biosurfactants, which are lipopeptides, glycolipids, fatty acids
(including phospholipids), polymeric and particulate biosurfactants36. Among these five classes
(see table 2), glycolipids and lipopeptides are the most popular classes, comprising most of the
biosurfactants that are being produced industrially and that are being used in a wide range of
applications. It was demonstrated that rhamnolipids, which belongs to the class of glycolipids,
have an excellent behaviour in removing petroleum derivatives and heavy metals37, enhancing
marine oil spill bioremediation38 and even to be used as a fungicide39. Others biosurfactants, like
sophorolipid and mannosylerythritol lipids are involved in plant protection due to their capacity of
inhibiting phytopathogenic fungi growth40. In general, these compounds have excellent properties
8
which allows their use in a variety of applications, even in food industry, preventing the formation
of biofilms in many household products, due to their antifungal and antibacterial activities41. In
fact, Rodrigues et al42 have shown that rhamnolipids from P. aeruginosa DS10-129 reduces the
adhesion of several bacteria and yeasts strain isolated from protheses explanted voice
prostheses to silicone rubber.
Table 2: Resume of the 5 classes of biosurfactants, including some examples of each class, and the
respectively microorganism producer. Table adapted from5
Class of biosurfactant Biosurfactant Microorganism producer
Glycolipids
Rhamnolipids P. aeruginosa
Pseudomonas sp.
Trehalolipids
R. erythropolis N.
erythropolis
Mycobacterium sp.
Sophorolipids T. bombicola T. apicola
T. petrophilum
Cellobiolipids U. zeae, U. maydis
Mannosylerythritol lipids M. rugulosa, M. aphidis and
M.antarctica
Lipopetides and
lipoproteins
Peptide-lipid B.licheniformis
Serrawetin S. marcescens
Viscosin P. fluorescens
Surfactin B. subtilis
Subtilisin subtilis B. subtilis
Gramicidinis B. brevis
Polymyxins B. polymyxa
Fatty acids and
phospholipids
Fatty acids C. lepus
Phospholipids T. thiooxidans
Polymeric Surfactants
Emulsan A.calcoaceticus
Biodispersan A. calcoaceticus
Mannan-lipid-protein A. calcoaceticus
Liposan C. lipolytica
Carbohydrate-protein-lipid D.polymorphis, P.
fluorescens
Protein PA P. aeruginosa
Particulate biosurfactants Vesicles and fimbriae A. calcoaceticus
Whole cells Variety of bacteria
9
2.2.2 Market assessment of microbial biosurfactants
Microbial biosurfactants have gained attention all over the world and nowadays there are
several companies selling microbial biosurfactants in the markets due their versatility in a wide
range of applications. In 2014, the market was evaluated in USD 13.5 million and the forecast to
2020 is to reach USD 17.5 million, with a CAGR (compound annual growth rate) of 4 % from 2014
until 202043. Regarding the amount of microbial biosurfactants produced, in 2014, approximately
150 tons were produced43.
Therefore, it is evident that biosurfactants have a huge potential to replace chemical
surfactants. Although, the industry of chemical surfactants and plant-derived biosurfactants
remains to be the world supplier of these compounds, due to several problems, including the
recover and purification of the product, high cost of raw materials and low yields in the production
41. So, if these problems were overcome, microbial biosurfactant production, probably, would
replace chemical surfactants in the markets41.
Nowadays, there are some companies supplying of biosurfactants, as described in table 3.
Table 3: Some of the largest microbial biosurfactant producing companies around the world. Adapted
from44
Company Location Product(s) Application(s)
TeeGene
Biotech UK
Rhamnolipids and
Lipopeptides
Pharmaceuticals, cosmetics,
and anti-cancer ingredients
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)
Groupe
Soliance France Sophorolipids Cosmetics
Henkel Germany
Sophorolipids,
Rhamnolipids,
Mannoslyerthritol lipids
Glass cleaning products,
laundry, beauty products
Evonik Germany Rhamnolipids
Sophorolipids Household products
Many of these companies are placed in Asia, Europe, and America. Figure 3 shows the
number of companies that produce microbial biosurfactant, in each country. In 2012, Europe was
on the top of microbial biosurfactants market, possessing 54.7 % of all market; consequently, it
10
was the region with more consumption of microbial biosurfactants. Then, the second region with
more importance in this market is United States of America, where it is expected to grow with a
CAGR of 5.6 % from 2014 to 202043. Pacific Asia is also growing in this market, although other
regions presented in surfactants market (such as Latin America) are not presented, due to the
high costs associated with the production of these microbial biosurfactants. Those facts explain
why the largest microbial biosurfactants producing companies are present in Europe and North
America.
Regarding the microbial surfactants used in the market, sophorolipids and rhamnolipids.
Sophorolipids, in 2012 had 54% of the microbial biosurfactants market. The market for MEL is
also growing, although there is only one industrial application, which is the production of a
cosmetic cream, containing MEL45
Figure 3: Compilation of the largest biosurfactant producer companies around the world, in each country.
Adapted from41
As described before, biosurfactants have a great potential in the world of surfactants. In figure
4, it is possible to observe the wide range of applications in many fields. Household products are
the major application of these compounds, due to their efficacy in detergency and the increasing
concerns regarding the toxicity of using chemical surfactants, so household products constituted
44,6% of biosurfactants sold in 2013.
Figure 4: Biosurfactants market volume share, by application in 201324.
0
1
2
3
4
5
6
7
8
China Japan SouthKorea
USA Germany Uk Belgium France
Asia America Europe
Nu
mb
er o
f co
mp
anie
s
Companies by country and continent
11
2.2.3 Renewable substrates and their use to produce biosurfactants
One of the main issues of biosurfactants is the price of the raw materials (e.g yeast extract
and glucose), as described in section 1.4.
Therefore, is essential to reduce the cost of the raw material, making a profitable process. For
that, most of the studies have been using renewable substrates to produce biosurfactants6. These
renewable substrates can be wastes of an industrial process, such as animal fat, residues of
vegetable oils. Those wastes are summarized in table 4.
Table 4: Summary of the several renewable substrates from industry with the potential to be used as a
carbon source. Adapted from6
Source industry Waste/residues as renewable substrates
Agro-industrial waste,
crops residues, animal fat
Bran, beet molasses, Bagasse of sugarcane straw of wheat,
cassava, cassava flour wastewater, rice, animal fat
Cofee processing
residues Coffee pulp, coffee husks, spent of free groundnut
Crops Cassava, potato, sweet potato, soybean, sweet sugar beet,
sorghum
Dairy industry Curd whey, milk whey, waste whey
Food processing industry Frying edible oils and fats, olive oil, potato peels rape seed
oil, sunflower, vegetable oils
Fruit processing industry Banana waste Pomace of apple and grape, carrot industrial
waste, pine apple
Oil processing mills
Coconut cake, canola meal, olive oil mill waste water, palm
oil mill, peanut cake, effluent, soybean cake, soapstock,
waste from lubricating oil
Among those renewable substrates, vegetable oils are the carbon sources most used to
produce biosurfactants. These oils are saturated compounds or unsaturated fatty acids with a
length of 16-18 carbon atoms, so it is a powerful component to be used as a carbon source,
leading to high productions of biosurfactant46,47.
Furthermore, there are others renewable sources that have been used as carbon sources to
produce biosurfactants, like dairy industry products, due to their high content in lactose and amino
acids and molasses (by product from sugarcane industry) containing a high content in sugar
compounds6,48. There are many reports showing the production of the same biosurfactant, but
with different renewable substrates. For example, it is demonstrated that biosurfactant
sophorolipid is produced using cheese whey49 Cryptococcus curvatus ATCC 20509 and Candida
bombicola ATCC 22214 ) and animal fat50 (Candida bombicola), as carbon source.
Lignocellulosic materials, one of the most promising renewable substrates due to his high
abundance on Earth47, can be used to produce bioethanol, fine chemicals, enzymes, pulp paper
and composites51,52. They are constituted by 10% to 25% of lignin, 20% to 30% of hemicellulose
and 40% to 50% of cellulose52. The hemicellulose component contains 15% to 35% of several
12
pentoses, hexoses and uronic acids53. Therefore, after the hydrolysis process of the
lignocellulosic material, the amount of sugar available as carbon source for a given microorganism
is substantial. Faria et al13 demonstrated for the first time the conversion of cellulosic materials
into mannosylerythritol lipids (glycolipid biosurfactant), showing that MEL can be produced using
lignocellulosic materials as a carbon source.
An important challenge for reduction of biosurfactants production costs are the selection of
new culture media, preferable using renewable substrates, and the design of new bioprocesses,
including downstream operations able to increase biosurfactants yield and reduce operation
costs. Complementary strategies could rely in strain engineering to improve microorganism
performance.
2.3 Mannosylerythritol Lipids (MEL)
2.3.1 Why MEL?
Mannosylerythritol lipids (MEL) is a biosurfactant belonging to the class of glycolipids (see
table 2) and possessing excellent properties to be used in a wide range of applications, especially
in cosmetic applications45. This biosurfactant is produced by a variety of microorganisms, being
mainly produced by fungi Ustilago maydis54 and Pseudozyma genus (see table 2), especially P.
antartica, P. rugulosa and P. aphidis. It is important to mention that these genus were renamed
to Moesziomyces, due to their close evolutionary taxonomy to Moesziomyces bulltus55.
MEL as represented in figure 5, contain 4-O-β-D-mannopyranosyl-meso-erythritol as the
hydrophilic group and fatty acids short-chains, as the hydrophobic group11. Accordingly to the
number and position of the acetyl group, MEL can have four forms: MEL-A, di-acylated
compound; MEL-B, mono-acylated compound in C6; MEL-C, mono-acylated compound in C4
and MEL-D, compound that deacylated36. There are other factors that influence the structure of
MEL, like the number of acylation in mannose and fatty acids length and their saturation.
.
Figure 5: Chemical structure of MEL and their types. MEL-A : di-acylated; MEL-B: mono-acylated in C6; MEL-C: mono-acylated in C456
There are several strains able to produce MEL and, accordingly to the type of strain, the
structure of MEL can be different. The strains that produce large quantities of MEL are M. aphidis,
13
M. rugulosa and M. antartica. These strains mainly produce MEL-A (about 70%), together with
MEL-B and MEL-C, although, the low water solubility of MEL-A limits the application of these
biosurfactant56.
Pseudozyma tsukubaensis is able to efficiently produce one type of MEL, MEL-B 57. Fukuouka
et al58 achieved the production of MEL-D, through an enzymatic reaction, removing the acetyl
groups. However, this year, Konishi et al59 developed a mutant of Pseudozyma hubeiensis SY16
to selectively produce MEL-D. Knowing that this specie (Pseudozyma hubeiensis SY16) mostly
produce MEL-C, the authors constructed a mat-1 knockout mutant, avoiding the formation of
acetylated forms of MEL (in this case, MEL-C), leading to the production of MEL-D (91.6 g/l).
It is important to know the composition of the MEL mixture produced, to better design the
downstream processing and applications.
2.3.2 Metabolic pathways for producing MEL
This section discusses the metabolic pathways for the conversion of MEL using vegetable oils
or sugars as carbon source.
By analysing the structure of MEL (figure 2), there are 3 main moieties present in the structure:
Mannose, erythritol and short-chain fatty acids. The assembly of these moieties to form MEL, is
regulated by a cluster of 4 genes, as described by Hewald et al 60. These genes are emt1, which
encodes for a mannosyltransferase, being responsible for the mannosylation of erythritol 61, mac
1 and mac 2, that encodes for an acetyltransferase, transferring short/medium fatty acids to C4/C6
hydroxyl groups present in mannose, and a last gene, mat-1, which encodes an acyltransferase,
leading to the acylation of MEL, forming MEL-A, MEL-B or MEL-C. (figure 6) 60,61.
Microorganisms usually degrade fatty acids by complete β-oxidation of fatty acids, forming
acetyl-coenzyme A (acetyl-coA). Acetyl-coA can be redirected to TCA cycle to produce ATP or
used to produce long-chain fatty acids that can be incorporated into cellular membrane.
Additionally, acetyl-coA can be used to elongate the fatty acids chain, forming long-chain fatty
acids. In case of fatty acids has a long chains, occurs an intact incorporation in cell membrane 62.
When the carbon source is glucose, three pathways are involved in the production of MEL,
such as pentose phosphate for producing erythritol, mannose and fatty acids. To produce
mannose, fructose-6-phosphate, from glycolytic pathway is converted into mannose-6-phosphate
by enzyme mannose-6-phosphate isomerase (EC 5.3.1.8), then mannose-6-phospahte is
converted into mannose-1-phosphate, reaction catalysed by GDP-D-mannose
pyrophosphorylase (EC 2.7.7.22)63. Erythritol is produced through the non-oxidative phase or
through oxidative phase of pentose phosphate pathway 63. Then, occurs the mannosylation of
erythritol, by mannosyltransferase (2.4.1). In the end of glycolysis, pyruvate is converted into
Acetyl-CoA by pyruvate dehydrogenase complex (EC 1.2.4.1), entering in TCA cycle. However,
to produce fatty acids, acetyl-coA reacts with oxaloacetate to form citrate, and only after high
concentration of this compound, citrate is translocated to cytoplasm where is cleaved, forming
oxaloacetate and acetyl-CoA, which enters in pathway of fatty acids biosynthesis 64. This system
is present only in oleaginous yeast.
14
When MEL is produced using vegetable oils as carbon source, the metabolism totally differs.
Initially, vegetable oils are cleaved by lipases forming glycerol and fatty acids (see figure 6). Then
partial β-oxidation of fatty acids will occurs, known as chain shortening pathway, as demonstrated
by Kitamoto et al65. Then the incorporation into mannosylerythritol occurs, leading to the
production of MEL63. Glycerol formed by the action of lipases, enter in glycolytic pathway, via
gluconeogenesis or allowing the formation of mannose and erythritol by the reactions described
before.
The following figure (figure 6), resumes the metabolic pathway to produce MEL from glucose
and soybean oil, described earlier.
Figure 6: Resume of possible metabolic pathways to produce MEL. Retrieved from66
2.3.3 Applications of MEL
MEL is one of the most promising biosurfactants, especially due to its properties in cosmetic.
Recent studies demonstrated that MEL-A can be used to treat skin injuries, showing moisturizing
activity67. In fact, a company from Japan (Toyobo Co., Ltd) has developed a cosmetic,
SurfMellow®, using MEL-A in its formulation45. Morita et al68 also tested if MEL can be used to
repair damaged hair, providing a smooth and flexible hair.
This biosurfactant also presents excellent properties in the field of medicine, by inducing cell
differentiation and apoptosis36. Some reports show that MEL induces the differentiation of human
promyelocytic leukaemia cell line HL60 69 and can down regulate tyrosine kinase in K562 cells,
inhibiting the cell proliferation and inducing differentiation 70. MEL also have anti-inflammatory
action, where it is demonstrated that can inhibit secretion of TNF-α, which is a tumour necrosis
factor, a cytokine involved in cell proliferation, differentiation, apoptosis and in a variety of
diseases 71. So, it is possible to say that there is a future in using MEL to treat diseases, although
more studies are required.
There are several reports showing the high affinity of MELs (MEL-A, MEL-B and MEL-C) in
binding to human immunoglobulin G (IgG) 72,73.
Nowadays, gene transfection is an emerging technology, where foreign nucleic acids are
transferred into another cell to produce a genetically modified cell. There are 3 different groups
15
of methods for gene transfection: biological, chemical and physical. The most used method is the
virus mediated-transfection, which belongs to the biological group, although there are lots of risks
associated with this method 74. Through the years, biosurfactans have gained importance in this
filed, due to their capacity in self-assembling (formation of micelles). Kitamoto et al 75 found that,
when MEL is dispersed in the water, has the ability to form giant vesicles. Moreover, MEL can
help in gene transfection, due to their high capacity in forming stable vesicles36. Inoh et al76
showed that MEL-A enhanced the efficacy of gene transfection by cationic liposomes and induce
the membrane fusion between cells and cationic-liposomes.
2.4 Fermentation processes to produce MEL
The typical medium described in the literature to produce MEL includes MgSO4, KH2PO4 and
yeast extract 11,54. However, considering the nitrogen source and carbon sources, several studies
have been performed to elucidate the most efficient compound capable of promoting high
concentration of MEL77.
2.4.1 Influence of carbon source in the production of MEL
As discussed in section 1.3, vegetable oils have been used as a carbon source to produce
biosurfactants. In the case of MEL, several vegetables oils were used, like castor oil 78 and
soybean oil79. Kitamoto et al8, tested six different vegetable oils at an initially concentration of 80
mL/L, where they demonstrated that the best vegetable oil to be used as a carbon source is
soybean oil, producing 34 g/l of MEL, using M. antarcticus T-34. Arévalo80 used M. antarcticus
PYCC 5048T and oil-based carbon sources: poultry oil (12.9 gMEL/l), waste frying oil (8.3 gMEL/l),
crude soybean oil (13.7 gMEL/l) and crude rapeseed oil (11.5 gMEL/l). In this study it was also tested
the production of MEL using lignocellulosic hydrolysate (Wheat straw) and glucose, obtaining 1.5
gMEL/l and 3.8 gMEL/l, respectively.
Considering the metabolic pathway to produce MEL, when soybean oil is used as a carbon
source, it is necessary to cleave the triglycerides, forming monoglycerides and being incorporated
into MEL (chain shortening pathway), therefore, in that way, Kitamoto et al 81, have produced MEL
using n-alkanes ranging from C12 to C18, and the highest yield (0.87 gMEL/l) was obtained using
6% (v/v) of n-octadecane. The microorganism used was M. antarcticus T-34.
Furthermore, there are studies showing the use of sugar compounds as carbon source
(xylose, glucose). Morita et al.12, showed that M. antarcticus T-34 is able to produce MEL in the
presence of glucose, where the maximum concentration of MEL obtained, using 40 g/l of glucose,
was 3.5 g/l. Consequently, when the authors used a fed-batch mode, supplying to the medium
120 g/l of glucose, each day, the final concentration obtained was 12 g/l.
Concerning the use of sugar compounds as a carbon source, there is a patent describing the
treatment of lignocellulosic residues by an enzymatic process, allowing the uptake of carbon
sources by microorganisms (M. aphidis; M. antarcticus), for the production of MEL10. The same
authors, reported the production of MEL, using pentoses (xylose and arabinose) as carbon source
for the first time11.
16
Beyond the use of lignocellulosic residues, other rich compounds were tested. Bhangale et
al82 have produced MEL using honey as a carbon source, and in this study, it was used the
microorganism M. antarcticus ATCC 32675 and the highest titre of MEL was 5.61 gMEL/L.
Dziegielewska and Adamczak83 have tested the production of biosurfactants (including MEL)
using renewable substrates. The best result was the use of soapstock and whey permeate or
molasses, where 90 g/l was obtained for M. aphidis and 40 g/l for M. antarcticus.
2.4.2 Influence of nitrogen source in the production of MEL
The complexity of each nitrogen source has the ability to interfere with metabolism of the cell,
by inducing several mechanism, such as biomass production84.
There are reports showing that the production of MEL occurred after nitrogen starvation9. Faria
et al11, performed a study where they compared the effects of the nitrogen source (using NaNO3),
at different days, in the production of MEL and biomass using three strains: M. aphidis, M.
antarcticus and M. rugulosa. In this studied, they used glucose and pentoses (xylose and
arabinose) as carbon sources. Therefore, they could conclude that a high ratio carbon/nitrogen
promoted the production of MEL, where the best result was the addition of carbon source (40 g/l)
and nitrogen source (3 g/l) at the beginning to the fermentation broth, leading to total consumption
of glucose and production of MEL (8 g/l).
Also, there are studies reporting that the nitrogen source can influence the type of MEL
produced. Rau et al14, using M. aphidis DSM 70725, tested different types of nitrogen source,
such as NaNO3, NH4Cl, NH4NO3 and (NH4)2SO4 with a concentration of 23.5 mM, and the best
result was the use of NaNO3, followed by NH4NO3, which did not result in a good result, since the
pH was not controlled and decreased until two. The authors also evaluated the production of MEL,
varying the ration of glucose/NaNO3 concentration, where they observe that the best result were
0/2 and 20/2, showing that high concentration of glucose and nitrate sodium led to lower yield of
MEL and high concentrations of SBO not consumed.
Kim et al79 used NH4NO3 to produce MEL and they observed that controlling the pH at 4, after
consumption of this nitrogen source, the production of MEL was improved.
Fan et al85, tested several types of medium, varying the nitrogen source (peptone or yeast
extract) and the presence of mineral salts (MgSO4.7H2O, KH2PO4 MnSO4, CuSO4). They have
showed that when the species M. aphidis ZJUDM3 is cultivated in a culture medium containing
peptone, manganese and copper salts, there are improvements in production of MEL-A, since
the manganese has the function to keep enzymes and proteins active.
2.4.3 Scale-up the production of MEL in bioreactors
Most of known biosurfactants are being produced in bioreactors (see table 3), which is the
case of surfactin and rhamnolipids. In fact, for produce rhamnolipids in bioreactors, the conditions
are well studied, and there is an industry well established around this biosurfactant.
Some attempts were performed using bioreactors to produce MEL using fed-batch of soybean
oil as carbon source. Rau et al9, using the microorganism M. aphidis DSM 14930, achieved 90
17
g/l of MEL with an yield of 0.86 gMEL/gsubstrate starting with 67 g/l of soybean oil and an addition of
37 g/l during three days. In the same study was obtained the highest yield reported in the literature
for MEL, 0.92 gMEL/gsubstrate, with a final titre of 165 g/l. This study started the fermentation with 17
g/l of soybean oil and 30 g/l of glucose, and, after 1.75 days, a concentrated solution was added
(glucose 285 g/l, sodium nitrate 16 g/l, yeast extract 14 g/l) was fed at 125 ml/h, and to control
the foam, 6.1 L were added to the medium during the first three days. However, this study
presents some limitations, since there is no information about how much SBO is used by the cell
and to control the foam.
Kim et al79, tested two types of fermentation: batch mode and fed-batch mode. In batch mode
they observed that MEL titre is improved from 37 g/l to 48 g/l, when the pH is controlled at 4 (since
they use NH4NO3 and after consumed the pH is reduced until 4). In fed-batch mode, it was
produced 95 g/l of MEL, but two pulses of soybean oil were added, 70 g/l and 100 g/l, at 50 and
100 hours respectively. In the end of the fermentation the concentration of residual oil was 20 g/l,
which will difficult downstream process to purify MEL.
2.5 Downstream processing of biosurfactants
Downstream processing comprise all the methods used to obtain and concentrate the final
product, constituting around 70% of the total costs in biological product86, therefore, it is important
to choose the best methods. Normally for downstream processing of biological products there are
3 stages: extraction, intermediary purification and ultra-purification. Although, depending on the
type of application, not all the stages are required.
When the biological product is intracellular, it is necessary to release the product off the cells
for further extraction of the product. So, before the extraction of the product, some methods are
required like centrifugation, to concentrate the cells, followed by cell disruption, allowing the
release of the product. In this category, sonication, homogenization, bead milling and osmotic
shock are the possible techniques to be used87.
The following step is to perform the extraction of biosurfactant, and, for these extraction, there
are several techniques that can be employed, such as acid precipitation; organic solvent
extraction; foam fractionation and crystallization88.
Acid precipitation is a technique that uses acid to precipitate biosurfactants, due to their
insolubility at low pH (which is not the case of MEL), as described in many studies. Although there
is another type of precipitation where the objective is to precipitate fatty acids presented in the
fermentation broth, a technique developed by Fleurackers89. This author described the separation
of sophorolipids from oleic acid using ions Ca2+ or Li+, which will bound to oleic acid, creating
soap. The best result was the use of the ion Ca2+, where 80% of oleic acid was removed as
insoluble calcium soap.
Organic solvent extraction uses the higher ability and partition of biosurfactants into organic
solvens (which is the case of MEL). These techniques (acid precipitation and organic solvent
extraction) allow, in one step, a high concentration of the biosurfactants. While acid precipitation
18
is a cheap method, organic solvent extraction is a method that can re-use the same solvent,
through a rotary evaporator40.
Due to the detergency capacity of biosurfactants, during the fermentation these compounds
are presented in the foam, so it was developed a technique, foam fractionation, allowing the
uptake of foam produced during the fermentation. This method involves the use of a device (foam
fractionation column) that can be integrated into the bioreactor, allowing the recovery of foam in
a continuous mode, leading to highly concentrations of bisourfactant, since only them are
presented in the foam90. In this technique, it is possible to extract 90-95% of the biosurfactant
produced in the fermentation.
After the extraction and concentration of the product it is necessary to retrieve the bio-products
produced during the fermentation. So, the choice of these techniques depends on the size, the
ionic charge (which is not the case of MEL, since it is a neutral molecule). Regarding the size and
the ionic charge, it can be used an ionic chromatography, due to the charge of biosurfactants,
allowing the adsorption of these compounds to the charged column. It can also be used size
exclusion chromatography, allowing the separation of biosurfactant from other products, based
on different size.
There are other methods like the adsorption using resins. This technique involves the use of
a hydrophobic resin, like XAD (hydrophobic copolymer of styrene-divinylbenzene resin), which
can interact with hydrophobic group of biosurfactants, allowing the separation of these
compounds from others. Normally, the organic solvent used to elute the biosurfactants in these
resins is ethyl acetate, a solvent also used in the organic solvents extraction. There is other type
of adsorption (adsorption on wood activated carbon). These methods of adsorption have the
advantage to re-use the same resin in recovery cycles, but is a method that involve high costs 91.
The final step of purification, used to achieve biological products with a high percentage of
purity, involves the use of normal or reverse phase, or even the use of ultrafiltration, microfiltration
and nanofiltration membrane. Membrane technology have been using increasingly, since it
presents several advantages, such as: high recovery yield, minimization of the denaturation and
degradation of the biological products desired to concentrate 92,93.
Considering these aspects, the challenge is to have a membrane able to discriminate between
products and impurities. Therefore, using membrane technology, some attempts were performed
to concentrate and purify biosurfactants, using their properties, such as the ability to form micelles,
known as CMC93,94. In this manner, Chen et al93 and Isa et al94 have developed a strategy to purify
surfactin, where they used a two-stage ultrafiltration. In first filtration, surfactin is retained since it
is in the form of micelles, and impurities are removed trough the permeate. Then, in the second
filtration, the micelles are disrupted and surfactin is removed to the permeate and impurities are
retained, obtaining a high purity (85%).
2.5.1 Downstream processing of MEL
There are few studies reporting separation and purification of MEL. Rau et al9 used several
techniques to separate MEL from fatty acids. They used multiple solvent extraction (three times
19
for each organic solvent), starting with MTBE (methyl tert-butyl ether), obtaining 75% of MEL,
15% of SBO and 10% of fatty acids. Then, this mixture was resolubilized in methanol and
extracted with cyclohexane, obtaining 91 % of MEL, 5 % of SBO and 4% of FA. At last,
cyclohexane was evaporated, and the mixture was extracted with a solution of n-hexane-
methanol-water (1:6:3). The organic solvents were evaporated, and the water was retrieved by
liophilization obtaining 100% of MEL. Although the authors have lost a lot of product, and, in the
end, only 8% of MEL remained. This low value is justified by the high difficulty in separating MEL
from fatty acids.
The same authors also tried to separate MEL using resins (XAD), which was unsuccessful
due to the absorption of fatty acids and soybean oil. They could achieve the best type of XAD
with high capacity for absorbing MEL with few fatty acids and resins.
Kitamoto et al95, developed a method to purify MEL using a silica gel column chromatography.
With this method, they achieved the separation of the four types of MEL (MEL-A, MEL-B, MEL-C
and MEL-D). After this, several papers reported this experience to purify MEL 96, 85.
Considering the ability of biosurfactants to form micelles, Andrade et al 97 have shown the
purification of MEL using ultrafiltration technology. In this study, they used cassava wastewater
(renewable substrate) to produce MEL, although using this substrate, a high amount of proteins
was also produced. Therefore, since MEL is capable of forming micelles, they have used a 100
kDa MWCO membrane, allowing the separation of proteins from MEL in one-step, obtaining a
purity of 80%.
20
Chapter 3 - Material and Methods
3.1 Materials
Reagents: Yeast extract (Oxoid), Malt extract (Oxoid), Peptone (Merck®), D-glucose
(Fischer), Agar (José M Vaz Pereira, S.A.), NaNO3 (LabKem), MgSO4 (Sigma-Aldrich®), KH2PO4
(Chem-lab), corn steep liquor, soybean oil (Olisoja), waste frying oil (McDonald´s), Lipase B
(Sigma).
Organic Solvents: Ethyl Acetate, Acetone, Hexane, Methanol, MTBE (tert-butyl methyl
ether), isopropanol, all from Fischer®; Acetyl Chloride from Fluka.
Equipment: Incubator (Memmert), Orbital shaker (Abalab, Agitorb 200), Bioreactor of 2.5L
(Infors: MT-minifors), Filtration dead-end cell (Sterlitech), rotary vapour (Buchi), centrifuge
(Sartorius 1-15P, Sigma, rotor 12000 rpm)
3.2 Microorganisms and maintenance
Moesziomyces yeast strains were provided by the Portuguese Yeast Culture Collection
(PYCC), CREM, FCT/UNL, Caparica, Portugal: M. antarcticus PYCC 5048T (CBS 5955), M.
aphidis PYCC 5535T (CBS 6821). These strains were plated in YM agar (yeast extract 3 g/l, malt
extract 3 g/l, peptone 5 g/l, D-glucose g/l and agar 20 g/l) and incubated for 3 days at 37°C.
From the plates, it was also prepared stock cultures of each specie (M. antarcticus and M.
aphidis). For that, each specie was grown up in liquid medium, and stored in 20%v/v glycerol
aliquots at -80ºC.
3.3 Media and Cultivation conditions
An inoculum was prepared by transferring the yeast colonies of M. antarcticus and M. aphidis
into an Erlenmeyer flask with 1/5 working volume of medium containing 3 g/l NaNO3, 0.3 g/l
MgSO4, 0.3 g/l KH2PO4, 1 g/l yeast extract, 40 g/l D-glucose, and incubated in the orbital at 27°C
with 250 rpm, for 48 h.
Batch cultivation: After 48h, 10% (v/v) of inoculum was added into an Erlenmeyer flask with
1/5 working volume of mineral medium (0.3 g/l MgSO4, 0.3 g/l KH2PO4, 1 g/l yeast extract,3 g/l
NaNO3 at initial pH 6.0) and 40 g/l of D-glucose as carbon source.
3.4 Shake flask cultivation
Aiming at increasing MEL titres and gain insights for optimization of MEL production in
bioreactors using optimized set of substrates, several fermentations were performed with M.
aphidis and M. antarcticus. Samples of 3 mL were taken at days 4, 7, 10 and 14 of fermentation,
to further analysis of MEL and FA in GC. For all the conditions tested, the fermentation started
with 10% (v/v) of inoculum added into an Erlenmeyer flask with 1/5 working volume of mineral
medium (0.3 g/l MgSO4, 0.3 g/l KH2PO4, 1 g/l yeast extract and 3 g/l NaNO3) for a total volume of
21
50 mL. Only glucose and soybean oil differed the concentration and feeding mode and the day it
was supplied to the medium.
Therefore, initially the production of MEL was tested using only soybean oil with several
concentrations of SBO (80, 60, 40 and 20 g/l) with the rest of the mineral medium described
earlier.
Then the production of MEL was tested using pulses of two carbon sources (hydrophilic and
hydrophobic). In this experience, all the conditions started with 40 g/l of glucose, and at the 4th
day of fermentation 40 g/l of glucose and concentrations of SBO ranging from 5 to 20 g/l were
added to the culture medium.
• [40glu,0:40glu,4]- Addition, at 4th day of 40 g/l of glucose (control)
• [40glu,0:40glu and 5sbo,4]- Addition, at the 4th day of glucose and 5 g/l of SBO
• [40glu,0:40glu and 10sbo,4]- Addition, at the 4th day of glucose and 10 g/l of SBO
• [40glu,0:40glu and 20sbo,4]- Addition, at the 4th day of glucose and 20g/l of SBO
To understand which carbon source should the fermentation start with, three conditions were
tested in biological duplicates:
• [40glu,0:40sbo, 4]- Starting the fermentation with 40 g/l of glucose and at the 4th day
a pulse of SBO 40 g/l were added to the medium
• [40sbo,0:40glu, 4]- Starting with 40 g/l of SBO and at the 4th day a pulse of 40 g/l of
glucose was added
• [40glu,0:20sbo,4]- Starting with 40 g/l of glucose and at the 4th day a pulse of 20 g/l
of SBO
A 3rd set of experiments was performed in duplicate: three compounds were tested (Corn steep
liquor, peptone, and yeast extract) in duplicate. At the beginning of the fermentation the medium
was composed by 40 g/l of glucose, 10 g/l of each compound tested (one at a time) and the others
mineral compounds described earlier. Then a pulse of 40 g/l of SBO was added to the medium
at 4th day. Since these compounds were able to produce high concentrations of biomass, it was
supplied to the medium 40 g/l of SBO at the day 14th of fermentation with the rest of the mineral
compounds, and the fermentation was extended until day 21 of fermentation.
A new fermentation assay was performed using waste frying oil (WFO). In this fermentation,
40 g/l of glucose was added to start the fermentation and at the 4th day 20 g/l of WFO was added
to the medium. This experiment was performed in duplicate.
3.5 Bioreactor cultivation
The experiments were performed in a 2.5 -L bioreactor, and filled with 1L of culture medium,
constituted by: 3 g/l NaNO3, 0.3 g/l MgSO4, 0.3 g/l KH2PO4, 1 g/l yeast extract, 40 g/l D-glucose
and 10% of inoculum, as described earlier. The agitation speed was set in a cascade mode
(between 150 rpm and 400 rpm), varying according to the dissolved oxygen, air flow was set to 1
vvm, temperature was controlled at 27ºC and the pH was not controlled.
22
During this experiment, two bioreactors were simultaneous used, testing two different
conditions in M. aphidis. For M. antarcticus it was only tested the condition of the 1st bioreactor.
The fermentation took 12 days and samples were taken every day for further analysis of MEL, FA
and dried-weight. For each strain, the fermentations were performed in biological duplicate.
• 1st bioreactor: The fermentation started with 40 g/l of glucose, and after the
appearance of foam (approximately 1 day) a pulse of 20g/l of soybean oil was added
aseptically to the medium to control the foam.
• 2nd bioreactor: The fermentation started with 40 g/l of glucose, and a pulse of 2g/l of
SBO was added aseptically to the medium for 10 days, in total of 20g/l.
Since in 1st bioreactor was added 20g/l of SBO at the 1st day, different of the condition
[40glu,1:20sbo,4] studied in shake flask, other set of fermentations in shake flask were created
starting with 40 g/l of glucose and the rest of the components and at the 1st day 20 g/l of SBO was
added to the medium.
3.6 Lipolytic assay
The enzymatic assays were performed as described by Gomes et al98. The substrate used for
the enzymatic assays was p-nitrophenyl butyrate. All enzymatic activities were carried out in a 96
well plate, and the reaction mixture was composed by: 2.63 mM of p-nitrophenol butyrate was
dissolved in 50 mM acetate buffer (pH 5.2) and 4% of triton-X-100.
To initiate the enzymatic assay, 90µL of p-nitrophenol butyrate 2.63 mM solution and 10 µL of
the supernatants was added. Then the reaction mixture was incubated at 37ºC for 15 minutes,
and after that, the reaction was stopped by adding 200 µL of acetone. The absorbance was
measured at 405 nm in a microplate spectrophotometer (MultiskanTM GO, ThermoFisher
Scientific), and the enzymatic activity was determined. One unit (U) of lipase activity is defined as
the amount of enzyme releasing 1 μmol p-nitrophenol per minute.
3.6.1 Enzymatic reaction using Lipase B (CAL-B)
An enzymatic reaction was performed to cleave triglycerides (TAG) from the aggregates
containing MEL and FA, extracted from the bioreactor using the microorganism M. aphidis, as
described in section 3.10.2. The reaction was performed by adding 0.1 g of the enzyme (CAL-B)
to 2 g/l of the aggregates extracted from the bioreactor, dissolved in 5 mL of phosphate buffer at
pH 7.0, during seven days at 37ºC.
3.7 Cell growth
Biomass was followed by measuring cell dried weight (CDW) at day 0, 1, 4, 7, 10 and 14 of
fermentation time. Cell dry weight was determined from 1 ml of culture broth by centrifugation at
13000 × g for 5 min, followed by cell pellet washing with 500 µL of deionized water (twice) and
drying at 60ºC for 48 h.
23
3.8 Sugar profile
Collected supernatants were diluted with sulphuric acid 0.05 M solution (1/2) and centrifuged
(Sartorius 1-15P, Sigma) at 13000 rpm for 1 min. Supernatants were collected and diluted with
sulphuric acid 0.05 M solution (1/10), resulting in a dilution of 1/20. The quantification was
performed by high performance liquid chromatography (HPLC), using a system Merck Hitachi,
Darmstadt, Germany) equipped with a refractive index detector (L-7490, Merck Hitachi,
Darmstadt, Germany) and an Aminex HPX-87H column (300 mm× 7.8 mm, Bio-Rad), at 50°C.
Sulfuric acid (0.005 M) was used as mobile phase at 0.4 ml/min.
3.9 Quantification of MEL
3.9.1 Methanolysis and GC analysis
During the fermentations, 1 or 3 mL of culture broth were periodically taken and freeze-dried.
The fatty acid content of the biological samples was determined by methanolysis and GC analysis
of methyl esters as described by Welz et al99.
Initially, pure methanol was cooled down to 0ºC under nitrogen atmosphere and acetyl chloride
was added under stirring over 10 minutes in the proportion of 20/1 (v/v), respectively. This
combination generated a water-free HCl/methanol solution. Culture broth samples, after freeze-
drying, were weighted and mixed with 2 ml HCl/methanol solution and 100 µL of internal standard,
4% (v/v) heptanoic acid an 96% (v/v) of n-Hexane. Then the samples were incubated for 1 h at
80°C for reaction into methyl esters.
The resulting product was extracted with 1 mL of n-hexane and 1 mL of water, then the organic
phase was retrieved and 1µL was injected in a GC system (Hewlett-Packard, HP5890), equipped
with a FID detector and a HP-Ultra 2 column. The oven was programmed from 140°C and
temperature raised to 170°C at 15°C/min, to 210°C at 40°C/min and to 310°C at 50°C/min. Final
time of 3 minutes. Nitrogen gas was used at a flow rate of 50 mL/h. MEL production is quantified
through the amount of C8, C10 and C12 fatty acids.
3.9.2 MEL extraction
To extract MEL from the fermentation in shake flask, 25 mL of ethyl acetate was added, an
equal volume of the fermentation broth retrieving the organic phase. This procedure was realized
three times. Then the organic phase was evaporated in a rotary evaporator, recovering ethyl
acetate and obtaining MEL.
MEL produced in bioreactor was extracted with approximately 500 ml of ethyl acetate
retrieving the organic phase and posterior evaporation in a rotary evaporator.
3.9.3 TLC analysis
Thin layer chromatography (TLC) was performed to evaluate the presence of MEL in a given
product, and the differences of MEL produced by two species: M. aphidis and M. antarcticus.
Aluminium plates (TLC-sheets Alugram Xtra SIL G/UV254) were cut with the dimensions of 8×4
cm with mobile phase consisting of: chloroform (CHCl3), methanol (MeOH) and water (H2O)
24
(65:15:2). Then the eluted compounds were revealed by heating the plate after spraying with a
solution of naphthol (1.5 g), sulfuric acid (6.5 mL), ethanol (51 mL) and water (4 mL).
3.10 Nanofiltration
MEL and FA obtained from the bioreactor (1st bioreactor) using the microorganism M. aphidis
were extracted as described in section 3.10.2.
In this section two types of membranes were used. The first membrane used 22% of
polybenzimidazoles (PBI) in a solution of dimethylacetamide with a MWCO distribution of 580 Da
to 540 Da. The second membrane used 17% of PBI in a solution of dimethylacetamide and it was
placed in a dead-end filtration cell.
The nanofiltration was performed using the aggregates (MEL and FA) solubilized in an organic
solvent with a total volume of 50 ml, with a concentration of 2 g/l. Initially it was tested, using the
membrane with 22% of PBI, ethyl acetate as an organic solvent at different pressures: 10, 20 and
30 bars. MTBE and isopropanol were also tested with 30 bar of pressure.
After this experiment, the membrane with 17% of PBI was tested, with a higher MWCO. For
this assay with the membrane, initially 50 mL of water and 50 mL of ethyl acetate were mixed,
and organic phase (with 3% of H2O) and aqueous phase were separated. For both phases,
MEL/FA were solubilized in a final concentration of 2 g/l. Then the filtration was performed at a
pressure of 8 bar.
For MEL, monoglycerides (MAG), diglycerides (DAG) and triglycerides (TAG) quantification,
the solutions were divided and evaporated for further analysis of MEL (see section 3.10.1) and
glycerides (see section 3.11.1).
3.10.1 Glycerides quantification
The concentration of monoglycerides (MAG), diglycerides (DAG) and triglycerides (TAG) in
the initial solution (feed), permeate and retentate were analysed by HPLC, as described by
Badenes et al100. The HPLC was equipped with a Chromolith Performance RP-18 endcapped
(100mm x 4.6mm x 2µm) column, an auto sampler (Hitachi LaChrom Elite L-2200), a pump
(Hitachi LaChrom Elite L-2130) and a UV detector (Hitachi LaChrom Elite L-2400) set up at 205
nm. The flow rate was set up at 1 ml/min and the injection volume was 20 µl. Three mobile phases
were employed: phase A consisted of 100% acetonitrile, phase B consisted of water 100% and
phase C comprising a mixture of n-hexane and 2-propanol (4:5, v/v). Quantification was carried
out using calibration curves of Glyceryl trioleate (~65 %, Sigma-Aldrich GmbH) for TAG, 1,3 –
Dioelin (≥99%, Sigma-Aldrich GmbH) and 1-oleoyl-rac-glycerol (≥99%, Sigma-Aldrich GmbH) for
MAG. 200 μL of each sample was retrieved and mixture with 1 μL of acetic acid 58.5 Mm and 799
μL n-hexane. Then it was centrifuged at 13000 rpm for 2 minutes, and the organic phase was
extracted and injected into the HPLC system.
25
Chapter 4 - Results and discussion
4.1 Studying the effect of using two carbon sources in the production of MEL
MEL, glycolipids with unique biosurfactant properties, are produced by Moesziomyces spp.
from different substrates, preferably vegetable oils101, but recently, within the iBB group, the use
of inexpensive lignocellulosic substrates has emerged as an attractive attempt to develop a more
sustainable process using renewable hydrophilic substrates, which also facilitate the downstream
processing, which is hampered when residual oils are present, common situation when vegetable
oils are used. Nevertheless, MEL titres achieved is relatively low posing difficulties to make the
process economically viable13.
The objective of this section was to achieve a condition with high titres of MEL with the
minimum concentration of residual fatty acids in the culture medium, obtaining MEL, at least with
a purity of 85% after simple ethyl acetate extraction. Reference soybean oil concentration (80 g/l)
was used for MEL production along with decreasing concentration of SBO to evaluate how the
MEL titre, productivity and residual FA are affected with initial SBO concentration (section 4.1.1).
Then, the three next sub-chapters explore how the simultaneous or intercalated utilization of
hydrophilic and hydrophobic carbon sources can improve MEL titres keeping low residual FA
(section 4.1.2 and 4.1.3). Along with the carbon source, alternative complex nitrogen sources
were used to study alternative means of improving MEL purity by either improving MEL titres
and/or reducing residual FA (Section 4.1.4).
4.1.1 MEL production using SBO
Most of the studies have used vegetable oils to produce MEL, obtaining relatively high titres
of MEL. In the literature, Kitamoto et al8 have obtained 34 g/l of MEL (yield of 0.48 gMEL/gsubstrate)
using M. antarcticus and 80 ml/l of SBO. In this case there is no information about the free fatty
acids presented in the medium. Rau et al14 obtained approximately 55 g/l with some SBO (around
3g/l) left in the medium to be consumed, using the same initial SBO (80 mL/L). In other study9,
the same authors tried to separate MEL from fatty acids using multiple solvent extraction,
obtaining 8% of MEL, as described earlier in section 2.5.1.
To test how MEL titre, productivity and residual FA are affected by initial SBO concentration,
the following initial SBO concentrations were considered for M. aphidis and M. antarcticus:
The assays were:
• Assay 1: 80 g/l of SBO
• Assay 2: 60 g/l of SBO
• Assay 3: 40 g/l of SBO
• Assay 4: 20 g/l of SBO
The characterization of the soybean oil used in these experiments is presented in annex 1.
In this set of fermentations, in the higher concentrations of SBO (80 and 60 g/l), some red
aggregates started to appear in the culture medium, in both strains with more relevance in M.
26
aphidis, a phenomenon also described by Rau et al9. Figure 7 represent one example of the
appearance of red aggregates.
To confirm that these aggregates were clusters of MEL, a TLC test was performed comparing
MEL directly extracted from fermentation broth and the red aggregates which are easy to collect
from M. aphidis cultures (figure 8) in which it is possible to observe the presence of both MEL and
residual fatty acids in the red aggregates. These bands were classified by comparison with a TLC
result obtained by Morita et al66.
From the result of TLC (figure 8), it is possible to observe the bands corresponding to fatty
acids, MEL-A. Considering MEL-B and MEL-C, it was not possible to identify by this analysis
which bands correspond to each type of MEL.
When red aggregates were present in culture, the concentration homogeneity in samples was
compromised. In that cases MEL was extracted from all broth and the results in figure 9 and 10
represent the concentration of MEL, FA and total biomass at the end of the fermentation (day 14).
In annex 2 the results of MEL, FA and biomass are represented, for the days 1,4,10 and 14 of
fermentation (data from non-homogeneous samples).
Figure 7: Images of culture medium for the condition 60 g/l of SBO in M. antarcticus at 14th
day of fermentation
Figure 8: Result obtained by TLC: a) Comparison of MEL extracted from M. antarticus and M. aphidis; b) Comparison between MEL extracted
from M. aphidis and aggregate from fermentation broth.
b) a)
MEL-C
MEL-C
27
Table 5: Summary of maximum MEL obtained, yield (product/substrate), yield in mol, purity factor and productivity for each condition with M. aphidis and M. antarcticus.
a) Final concentration of MEL and FA in the fermentation broth
b) Yield (Titre of MEL produced/ Concentration of carbon source added)
c) Yield (mol of carbon in MEL produced/ total mol of carbon added)
d) Purity factor (MEL produced/ (MEL produced + concentration of fatty acids))
Strain Condition MEL
a) (g/l)
FAa)
(g/l)
Yield b)
(p/s)
Yield c)
(p/s) mol
Purity
d) Factor
Productivity
e) (g/l.h)
M.a
ph
idis
[80g/l SBO] 21.79 26.62 0.272 0.063 0.45 0.0649
[60g/l SBO] 18.71 8.13 0.312 0.072 0.70 0.0557
[40g/l SB0] 13.24 2.38 0.331 0.077 0.85 0.0394
[20g/l SB0] 10.07 1.84 0.503 0.117 0.85 0.0300
M.a
nta
rcti
cu
s [80 SBO g/l] 19.00 32.23 0.238 0.054 0.37 0.0565
[60 SBO g/l] 18.06 11.11 0.301 0.078 0.62 0.0537
[40 g/l SB0] 14.00 0.86 0.350 0.091 0.94 0.0417
[20 g/l SB0] 10.00 0.49 0.500 0.129 0.95 0.0298
0
5
10
15
20
25
30
35
[80g/L SBO ] [60g/L SBO ] [40g/L SBO] [20g/L SBO]
[Tit
re]
g/L
Figure 9: Maximum production of MEL (White bars), fatty acids (black bars) and cell dry weight (grey bars) in each condition (80, 60, 40 and 20g/l of SBO) for M.
aphidis.
0
5
10
15
20
25
30
35
[80g/L SBO] [60g/L SBO ] [40g/L SBO] [20g/L SBO]
[Tit
re]
g/L
Figure 10: Maximum production of MEL (White bars), fatty acids (black bars) and biomass (grey bars) in each condition (80, 60, 40 and 20g/l of SBO) for M.
antarcticus
28
In both M. aphidis and M. antarcticus, it is possible to observe that higher SBO concentrations
(80 and 60 g/l) gave rise to higher MEL titres of MEL (>18 g/l), although the amount of fatty acids
is also high, with a ratio of MEL:FA lower than 3/1, which means a MEL purity lower than 85%.
Nevertheless, at the lower concentrations of SBO (40 and 20 g/l), the amount of fatty acids were
lower, below 2.5 g/l and a final MEL purity higher than 85%. Cell dry weigh was similar between
the high concentrations of SBO (80 and 60 g/l) and then decreased as the concentration of SBO
decreased.
In table 5 it is possible to compare MEL and FA titres, yields and purity factor mentioned earlier
𝑃𝑢𝑟𝑖𝑡𝑦 𝑓𝑎𝑐𝑡𝑜𝑟 =𝑀𝑎𝑥𝑖𝑚𝑢𝑚 𝑜𝑓 𝑀𝐸𝐿 𝑝𝑟𝑜𝑑𝑢𝑐𝑒𝑑
𝑀𝑎𝑥𝑖𝑚𝑢𝑚 𝑀𝐸𝐿 𝑝𝑟𝑜𝑑𝑢𝑐𝑒𝑑+𝑀𝑎𝑥𝑖𝑚𝑢𝑚 𝑜𝑓 𝑓𝑎𝑡𝑡𝑦 𝑎𝑐𝑖𝑑𝑠 𝑎𝑐𝑐𝑢𝑚𝑙𝑎𝑡𝑒𝑑 (equation 1)
The yield obtained for condition 20g/l SBO is higher (0.5 gMEL/gsubstrate) than the value obtained
by Kitamoto et al8 (0.48 gMEL/gsubstrate). However, the final concentration of MEL is not so high.
4.1.2 Pulses of two carbon sources (hydrophilic and hydrophobic) to increase MEL
titres.
The use of lignocellulosic-based sugars for MEL production has been reported as an
interesting alternative to vegetable oils due to: renewability and low price of materials; facilitated
downstream conferred by the hydrophilic properties of sugars and low level of residual fatty acid.
However, the utilization of glucose and/or xylose to produce MEL is reported to lead to low yields
(approximately 0.1 gMEL/gsubtrate)11,12.
Thus, the present section aims to evaluate the utilization of a hydrophobic carbon source, in
addition to the hydrophilic carbon source, evaluating if it is possible to increase MEL yield and
maintaining a low accumulation of fatty acids in the culture medium. Since MEL is formed by a
hydrophilic and hydrophobic group, the idea is to observe if feeding the medium with glucose
(hydrophilic source) and soybean oil (hydrophobic source) could increase MEL titres. In this
experience, fed-batch fermentations were performed. Based culture condition included the
utilization of 40 g/l of glucose and a feed pulse at day 4 of 40 g/l of glucose ([40glu,0:40glu,4]).
Alternative feeding strategy included, in addition to 40 g/l of glucose: 5 g/l of soybean oil ([40glu,0:
40glu and 5sbo,4]); 10 g/l of soybean oil ([40glu,0:40glu and 10sbo,4]); and 20 g/l of soybean oil
([40glu,0:40glu and 20sbo,4]).
29
Observing figure 11 a), it is possible to observe that a pulse of 40 g/l of glucose and 5 g/l of
SBO has no positive influence in the production of MEL when compared with the condition with a
pulse of 40 g/l of glucose at the end of the fermentation, since MEL increased between day 4 and
7 to 2.7 g/l, higher than the 0.9 g/l observed in the control. However, this difference decreased
after day 7 with a decrease in MEL concentration in the [40glu,0: 40glu and 5sbo,4] conditions.
When the SBO feed concentration increased to 10 g/l and 20 g/l, an increase of MEL production
0
4
8
12
16
20
0 2 4 6 8 10 12 14
ME
L T
itre
(g/L
)
Time (days)
a)
0
4
8
12
16
20
24
0 2 4 6 8 10 12 14
Fatty a
cid
s c
onsum
ption
(g/L
)
Time (days)
b)
0
5
10
15
20
25
30
0 2 4 6 8 10 12 14
Dry
weig
ht
(g/l)
Time (days)
c)
0
4
8
12
16
20
0 2 4 6 8 10 12 14
ME
L t
itre
(g/L
)
Time (days)
d)
0
4
8
12
16
20
24
0 2 4 6 8 10 12 14
Fatty a
cid
s c
onsum
ption
(g/L
)
Time (days)
e)
0
5
10
15
20
25
30
0 2 4 6 8 10 12 14
Dry
weig
ht
(g/L
)
Time (days)
f)
Figure 11: Production of MEL (a), consumption of FA (b), formation of biomass (c) by M. aphidis; Production of MEL (d), consumption of FA (e), formation of biomass (f) by M. antarcticus for the conditions:
[40glu,0:40glu4] (Dashed line with ); [40glu,0:40glu and 5sbo,4] (Line with▲); [40glu,0:40glu and 10sbo,4] (Dashed line with ●) and [40glu,0:40glu and 20sbo,4] (Line with ■). Red markers means the
presence of red aggregates in culture medium.
30
was observed, which could indicate a preference of M. aphidis for this carbon source. Even
though, the production of MEL in both conditions, [40glu,0:40glu and 10sbo,4] and [40glu1:40glu
and 20sbo,4] is quite similar. Such results can be explained by the appearance of red aggregates
in condition [40glu,0:40glu and 10sbo,40], represented in figure 12, after the day seven of
fermentation. Figure 12 represents red aggregates at days 7, 10 and 14 in the condition of 40 g/l
of glucose and 20 g/l of soybean oil, justifying the lower MEL concentration observed after day 7
of fermentation (due to non-homogeneous sampling), and why this condition ended the
fermentation with the same amount of MEL and FA than the condition [40glu,0:40glu and
10sbo,4]. To retrieve the final value of MEL and FA of the condition [40glu,0:40glu and 10sbo,40]
the culture medium was completely extracted, and a total of 10.6 g/l of MEL and 3.4 g/l of FA
were obtained, which is higher than the amount of MEL (6.5 g/l) obtained for the condition
[40glu,0:40glu and 10sbo,4].
As described for M. aphidis, the strategie [40glu,0:40glu,4] and [40glu,0:40 glu and 5sbo,4]
have a similarly behaviour in producing MEL by M. antarcticus for 14 days of fermentation, in
which no differences were observed when 5 g/l of SBO were used (figure 11c). The conditions
[40glu,0:40 glu and 10sbo,4] and [40glu,0:40 glu and 20sbo,4] led to an improvement on MEL
production from around 5 g/l to 9.6 g/l and 16.0 g/l respectively, higher values than those observed
in M. aphidis in the same culture conditions.
Regarding the fatty acids profile (figure 11b) it is possible to observe that all the conditions
tested ended the fermentation with the same amount of fatty acids in the culture medium,
approximately 2 g/l for M. aphidis and below 2 g/l for M. antarcticus.
Analysing the results for biomass production (figure 11 c and e), the behaviour of the two
species is almost identically, with the difference that M. antarticus is more able to produce more
biomass.
c) a) b)
Figure 12: Evolution of the red aggregates in M. aphidis cultivation for the condition [40glu,0:40glu and 20sbo,4] at: a) day 7; b) day 10; c) day 14 of fermentation
31
Figure 13: Typical behaviour for glucose consumption for M.aphidis (grey line) and M.antarcticus (black line)
Figure 13 portrays a typical glucose consumption profile for glucose and respective feed
strategy, at 40 g/l concentration.
Analysing the yields for each condition in each strain (table 6), it is possible to observe that
increasing the concentration of oil added at day 4 of fermentation, increases, not only MEL titres,
but also MEL yields, pointing out that the oil added improve the culture efficiency to transform the
carbon from the substrate into the product, MEL. Nevertheless, this effect is observed only when
oil at a concentration of 10 g/l or more is added to the cultivation.
In M. aphidis, the higher yield of [40glu1:40glu and 20sbo,4] condition is observed only after
an extraction of all culture broth due to non-homogeneous sampling derived from the large red
aggregates formed.
Comparing the two strains, it is possible to observe that M. antarcticus, regardless the
strategie, produced higher MEL titres and yields, as well as higher purity ratio.
Morita et al12 obtained 12 g/l of MEL (with an yield of 0.033 gMEL/gsubstrate) using M. antarcticus
T-34. The fermentation started with 120 g/l of glucose and every 7 days, a feed of 120 g/l was
supplied to the medium during 21 days. Comparing to the results here obtained, it corroborates
that the combination of hydrophilic and hydrophobic carbon sources increases MEL yields if
compared with large glucose addition to the cultivation.
0
5
10
15
20
25
30
35
40
45
0 2 4 6 8 10 12 14
Glu
cose c
onsum
ption (
g/l)
Time (days)
32
Table 6: MEL and FA titres, yields, purity factor and productivities in 14 days fed-batch cultivation of M. antarcticus and M. aphidis. The condition marked at bold, represents the extraction of MEL and FA from
the all broth.
a) MEL and FA produced at day 14th of fermentation
b) Yield (Titre of MEL produced/ Concentration of carbon source added)
c) Yield (mol of carbon in MEL produced/ total mol of carbon added)
d) Purity factor (MEL produced/ (MEL produced + concentration of fatty acids))
4.1.3 Development of a fed-batch strategy for M. aphidis and M. antarcticus cultivation
using hydrophilic and hydrophobic carbon source
In the previous chapter, it was shown that a pulse of two carbon sources (hydrophilic and
hydrophobic) can improve MEL production without almost no accumulation of FA in the culture
medium. However, it seems that the yields and productivity for both species are very low, even in
the condition where 20g/l of oil was added to the medium at 4th day of fermentation, and it is not
clearly which carbon source should the fermentation start.
Since, in the literature, MEL has been mostly produced using vegetable oils77,79,9 in the
beginning of the fermentation, this chapter tries to understand if the order or carbon source affect
MEL production, and how these two carbon sources can be combined to improve MEL titres,
maintaining a low accumulation of fatty acids.
So, three conditions were tested: start cultivation on 40 g/l of glucose and pulse feeding of 20
g/l of SBO at day 4 ([40glu,0:20sbo,4]) or 40 g/l of SBO ([40glu,0:40sbo,4]); and start cultivation
on 40 g/l of SBO and a pulse feeding at day 4 of 40g/l of glucose ([40sbo,0:40glu,4]). The results
are represented in figure 14.
Strain Condition MEL
a) (g/l) FA a) (g/l)
Yield b) (p/s)
Yield c) (p/s) mol
Purity d)
Factor Productivity
e) (g/l.h)
M.aphidis
[40glu1:40glu4] 2.98 1.72 0.037 0.017 0.634 0.009
[40glu1:40 and 5sbo,4]
1.04 1.22
0.012 0.005 0.461 0.003
[40glu1:40glu and 10sbo,4]
6.03 1.78
0.067 0.028 0.772 0.018
[40glu1:40glu and 20sbo,4]
6.53 1.65
0.065 0.026 0.798 0.019
[40glu1:40glu and 20sbo,4]
10.58 3.39
0.106 0.042 0.757 0.031
M.antarcticus
[40glu1:40glu4] 4.67 0.67 0.058 0.028 0.874 0.014
[40glu1:40 and 5sbo,4]
3.16 0.97 0.037 0.017 0.765 0.009
[40glu1:40glu and 10sbo,4]
9.64 0.76 0.107 0.046 0.927 0.029
[40glu1:40glu and 20sbo,4]
16.03 1.91 0.160 0.065 0.893 0.048
33
0
10
20
30
40
50
0 2 4 6 8 10 12 14
Dry
weig
ht
(g/L
)
Time (days)
c)
0
10
20
30
40
50
0 2 4 6 8 10 12 14
Glu
cose c
onsum
ption
(g/L
)
Time (days)
d)
0
5
10
15
20
25
30
0 2 4 6 8 10 12 14
ME
L t
itre
(g/L
)
Time (days)
a)
0
10
20
30
40
50
0 2 4 6 8 10 12 14
Fatty a
cid
s c
onsum
ption
(g/L
)
Time (days)
b)
0
5
10
15
20
25
30
0 2 4 6 8 10 12 14
ME
L t
itre
(g/L
)
Time (days)
e)
0
10
20
30
40
50
0 2 4 6 8 10 12 14
Fatty a
cid
s c
onsum
ption
(g/L
)
Time (days)
f)
0
10
20
30
40
50
0 2 4 6 8 10 12 14
Dry
weig
ht
(g/L
)
Time (days)
g)
0
10
20
30
40
50
0 2 4 6 8 10 12 14
Glu
cose c
onsum
ption
(g/L
)
Time (days)
h)
Figure 14: Production of MEL (a), consumption of FA (b), formation of biomass (c) and glucose consumption (d), by M. aphidis; Production of MEL (e), consumption of FA (f), formation of biomass (g) and glucose consumption (h), by M. antarcticus for the conditions: [40sbo,0:40glu,4] (Dashed line with
▲); [40glu,0:20sbo,4] (Line with ●) and [40glu,0:40sbo,4] (Line with ■). Red markers represent the presence of red aggregates in cultivation.
34
By analysing the results obtained for M. aphidis (figure 14a), it is possible to observe higher
MEL titres in the condition [40glu,0:40sbo,4], 17.6 g/l, when compared to condition
[40sbo,0:20sbo,4], 12.4 g/l. For the condition [40glu,0:40sbo,4] it appeared red aggregates,
justifying the reduction of MEL from day 7 until day 10 of fermentation.
Both conditions [40glu,0:20sbo,4] and [40sbo,0:40glu,4] reaches the same tire of MEL (around
12g/l) and in the first condition the total amount of carbon is lower. However, when it is used only
40 g/l of SBO in the beginning, the production of MEL is 13 g/l (figure 9), a value of MEL a bit
higher than the values obtained for [40glu,0:20sbo,4] and [40sbo,0:40glu,4]. This seems to prove
that M. aphidis has a high preference for produce MEL using SBO, and glucose, almost has no
influence in MEL production.
For M. antarcticus (figure 14e), it was observed higher MEL titres in condition
[40glu,0:40sbo,4], 24.7 g/l, than the condition [40sbo,0:20sbo,4], 18.3 g/l. Comparing the two
strains, it is possible to observe that M. antarcticus, regardless the condition, produced higher
MEL titres and yields, as well as higher purity ratio. For both species, the condition
[40sbo,0:20sbo,4] ends the fermentation with lower concentration of fatty acids than the condition
[40glu,0:40sbo,4] (see table 8), and so, differences are observed in purity factor.
Considering the glucose consumption in M. antarcticus (figure 14h) and M. aphidis (figure
14d), both have a similar behaviour in the two strains, with virtually all glucose consumed within
4 days (when added at day 0), and until day 10 when glucose was added at day 4. For biomass
production (figure 14d and f), the conditions [40glu,0:40sbo,4] and [40glu,1:20sbo,4] have the
same behaviour, which means that the same amount of SBO and glucose were used to produce
biomass and other components of the cell. The condition [40sbo,0:40glu,4], in M. antarcticus, has
produced more biomass than the other conditions, but, for M. aphidis the production was higher
in the first four days of fermentation, and after day 4, biomass production was similar between the
conditions.
By analysing table 8, it is possible to observe that in both strains, the condition
[40glu,0:40sbo,4] has the highest yield, and the condition [40sbo,0:40glu,4] have the lowest yield,
showing that the feed of glucose at day 4 of fermentation does not improve MEL yields. For M.
aphidis is more clear, since using 40 g/l of SBO, the value of MEL is a bit higher than the
conditions [40glu,0:40glu,4] and [40glu,0:20sbo].
For all the conditions, M. antarcticus has produced more MEL than M.aphidis, which reflects
in a purity factor higher, even for conditions where FA is equally between the species. So, it seems
that for M. antarcticus the combination of carbon sources (hydrophilic/hydrophobic) have led to
higher titres of MEL than M. aphidis.
35
Table 7: Summary of maximum MEL obtained, yield (product/substrate), yield in mol, purity factor and
productivity for each condition in M. aphidis and M. antarcticus.
a) MEL and FA produced at day 14th of fermentation
b) Yield (Titre of MEL produced/ Concentration of carbon source added)
c) Yield (mol of carbon in MEL produced/ total mol of carbon added)
d) Purity factor (MEL produced/ (MEL produced + concentration of fatty acids))
Kitamoto et al8 reported a MEL production of 34 g/l using Candida antartica T-34 with 8% of
SBO (approximately 70 g/l), obtaining an yield of 0.48 gMEL/gsubstrate, higher than the best yield
obtained for M. antarticus in this work (0.3 gMEL/gsubstrate). Although the authors have used high
concentration of SBO, and as discussed earlier (chapter 4.1.1), these conditions lead to the
accumulation of fatty acids in the culture medium.
Arévalo80 reported a MEL production of 18.3 g/l with 70 g/l of SBO and using the same strain
of the present work (M. antarticus PYCC 5048T). The yield obtained (0.26 gMEL/gsubstrate) is lower
than the yield obtained for the condition [40glu,0:40sbo,4].
Rau et al14 tested several conditions, using the microorganism M. aphidis DSM 70725, where
one of them was started with 70 g/l of SBO and added mannose (40 g/l) and erythritol (40 g/l) at
different days. The best result was obtained by adding mannose or erythritol after the cell reached
stationary phase (day two of fermentation), obtaining 70 g/l of MEL with a yield of 0.58
gMEL/gsubstrate, which is higher than the yield obtained for the condition [40glu,0:40sbo,4], 0.22
gMEL/gsubstrate.
4.1.4 Producing MEL using compounds enrichment with nitrogen
In the previous chapter, the best strategie to achieve high titres of MEL was the condition
starting with 40 g/l of glucose and at day 4 a pulse of 40 g/l of SBO ([40glu,0:40sbo,4]). Although,
in both species, this condition ended with a considerable amount of fatty acids, with a purity factor
of 0.649 for M. aphidis and 0.755 for M. antarcticus. Since these values are a little far from the
threshold defined in the beginning (at least 85% of MEL purity), it was tested the media
enrichment using organic nitrogen sources, to observe if they can stimulate the consumption of
fatty acids and the increase of MEL production.
For that, it was used 3 different compounds: peptone (10 g/l), corn steep liquor (10 g/l) and
yeast extract (10g/l). All these fermentations started with 40g/l of glucose and at day 4, 40 g/l of
Strain Condition MEL a)
(g/l) FA
(g/l) a)
Yield b) (p/s)
Yield c) (p/s) mol
Purity d) Factor
Productivity e) (g/l.h)
M. aphidis
[40glu,0:40sbo,4] 17.61 9.54 0.220 0.073 0.649 0.052
[40sbo,0:40glu,4] 12.40 3.47 0.155 0.052 0.781 0.037
[40glu,0:20sbo,4] 11.59 3.00 0.193 0.073 0.794 0.035
M. antarcticus
[40glu,0:40sbo,4] 24.69 8.00 0.309 0.104 0.755 0.074
[40sbo,0:40glu,4] 18.65 1.96 0.233 0.078 0.905 0.056
[40glu,0:20sbo,4] 14.84 1.47 0.247 0.094 0.910 0.044
36
SBO were supplied to the medium (condition [40glu,0:40sbo,4]) and the results are presented in
figure 15.
Since these three compounds are able to produce high concentrations of biomass at day 14
(figure 15c and 15g), another pulse of 40 g/l of SBO was added and the fermentation was
extended until day 21, in order to observe if MEL production would increase after the cells reached
the maximum concentration of biomass (approximately 50 g/l).
By analysing the values obtained for each condition in M. aphidis and comparing them with
the condition control (see figure 15a), it is possible to confirm that none of the three compounds
tested have improved MEL production, since all the three compounds have led to the production
of high concentrations of biomass (figure 15d). It seems that these compounds have redirected
carbon source to the formation of biomass, leading to higher concentrations of biomass (figure
15c and g). Even though, MEL produced in the conditions CSL and yeast extract, have reached
14.7 g/l and 10.0 g/l respectively. Although, for the condition using peptone, there was low
production of MEL, and after the addition of 40 g/l of SBO at 14, fatty acids decreased until day
17, but not consumed afterwards.
For the conditions using corn steep liquor (CSL) and yeast extract, the biomass have reduced
from day 17 to day 21 of fermentation.
Observing the results for M. antarcticus (figure 15e), there is no further MEL production after
SBO addition at day 14. For this strain, peptone and yeast extract have stopped the production
of MEL after day 10 of fermentation and\, although a low residual amount of FA in cultivation,
MEL titres observed are lower to the control (9.1 g/l, figure 14e). When CSL was used it is possible
to observe a significant increase in MEL production from day 10 to 14 (reaching 14.85 g/l).
37
0
10
20
30
40
50
0 2 4 6 8 10 12 14 16 18 20
Fatty a
cid
s c
onsum
ption
(g/L
)
Fermentation (days)
b)
0
10
20
30
40
50
0 2 4 6 8 10 12 14 16 18 20
Glu
cose c
onsum
ption
(g/L
)
Fermentation (days)
d)
0
10
20
30
40
50
60
70
80
0 2 4 6 8 10 12 14 16 18 20
Dry
weig
ht
(g/L
)
Fermentation (days)
c)
0
10
20
30
40
50
0 2 4 6 8 10 12 14 16 18 20
Fatty a
cid
s c
onsum
ption
(g/L
)
Fermentation (days)
f)
0
10
20
30
40
50
0 2 4 6 8 10 12 14 16 18 20
Glu
cose c
onsum
ption
(g/L
)
Fermentation (days)
h)
0
10
20
30
40
50
60
70
80
0 2 4 6 8 10 12 14 16 18 20
Dry
weig
ht
(g/L
)
Fermentation (days)
g)
0
5
10
15
20
25
0 2 4 6 8 10 12 14 16 18 20
ME
L t
iitre
(g/L
)
Fermentation (days)
e)
0
5
10
15
20
25
0 2 4 6 8 10 12 14 16 18 20
ME
L t
itre
(g/L
)
Fermentation (days)
a)
Figure 15: Production of MEL (a), consumption of FA (b), formation of biomass (c) and glucose consumption (d) by M. aphidis; Production of MEL (e), consumption of FA (f), formation of biomass (g)
and glucose consumption (h) by M. antarcticus for the conditions: peptone (Dashed line▲); yeast
extract (Dashed line with ●) and corn steep liquor (Line with ■).
38
In both species, none of the compounds has increased MEL production, more than the control
([40glu,0:40sbo,4]), maybe, because all of them have the ability to redirect carbon source for the
formation of biomass. Also, the yields are lower than the control (table 8).
The productivity is also lower (0.038 g/l.h), when compared to the control (0.052 g/l.h).
Therefore, these compounds in high concentrations seems to stimulate the biomass production.
Considering the results for CSL, in M. aphidis, the amount of fatty acids at day 14 was similar
to the amount of fatty acids in the control ([40glu,0:40sbo,4]), however the production of MEL was
lower in the case of CSL. These results with enriched media may point out that some nutrient
limitation might be needed for MEL production. For instance, the C/N (carbon to nitrogen) ratio
was lower in this section when compared with previous ones11.
Table 8: Summary of MEL obtained at day 14th of fermentation, yield (product/substrate), yield in mol,
purity factor and productivity for peptone, CSL yeast extract and the control ([0:40sbo] with the normal components) for M. aphidis and M. antarcticus.
a) MEL and FA produced at day 14th of fermentation
b) Yield at 14th day (Titre of MEL produced/ Concentration of carbon source added)
c) Yield at 14th day (mol of carbon in MEL produced/ total mol of carbon added)
d) Purity factor (MEL produced/ (MEL produced + concentration of fatty acids))
Konishi et al103 have found that 10 g/l is the best concentration of yeast extract to improve MEL
production in P. hubeiensis SY62, using 50 g/l of olive oil and 50 g/l of glucose. Kitamoto et al8,
have also tested the effect of CSL (2%) and yeast extract (0.05%) using M. antarcticus, however
did not improve MEL production when compared to the control. The authors also observed an
increase in MEL production from yeast extract to CSL, in line with observations of figure 15e.
Considering the results for CSL, in both species, it is possible to observe that the yields
obtained were higher compared to yeast extract and peptone. Even though, it seems that CSL is
inhibiting MEL production. Since CSL is constituted by 40% of proteins, 21% of lactic acid, 16%
of nitrogen free extract, and rich in vitamins and aminoacids104, maybe lactic acid is interfering
with MEL production, explaining why MEL production in high concentrations, only occurs after
day 10 of fermentation in M. antarcticus (figure 15e).
Sharma et al104 have shown that 10% of CSL inhibited the production of pullulan
(exopolysaccharide) and with 5% of CSL was the best result. This means that the key factor
Strain Condition MEL a) (g/l) FA a)
(g/l)
Yield b)
(p/s)
Yield c) (mP/mS)
Purity d)
Factor
Productivity e) (g/l.h)
M. aphidis
CSL 9.20 9.77 0.115 0.038 0.471 0.027
Yeast extract
8.00 1.74
0.100 0.033 0.821 0.024
Peptone 2.33 1.35 0.029 0.009 0.633 0.007
Control 17.61 9.54 0.220 0.0732 0.649 0.052
M. antarcticus
CSL 14.85 3.06 0.186 0.061 0.829 0.044
Yeast extract
4.56 5.70
0.057 0.019 0.864 0.014
Peptone 0.68 1.21 0.009 0.003 0.415 0.002
Control 24.69 8.00 0.309 0.1035 0.755 0.074
39
should be the concentration of CSL, as discussed earlier, and so, more studies are required to
find the optimal concentration of CSL to be used in the fermentation process.
4.1.5 Lipolytic activity
As described earlier (see chapter 2.3.2- Metabolic for producing MEL), after oil addition, the
first step of the cell is the cleavage of triglyceride (TAG) molecule forming glycerol and fatty acids
chain, and then, glycerol, which is converted to glycerol-3-phosphateby glycerol-kinase (2.7.1.30)
enters in glycolytic pathway and fatty acids chains are partially β-oxidized.
In that way, lipase activity was measured for the conditions: [80g/l SB0], [40glu,0:40glu,4],
[40glu,0:40sbo,4] and [40sbo,0:40glu,4] for both species.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
0 2 4 6 8 10 12 14
Lip
oytic a
ctivity
(IU
/mL)
Time (days)
a)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0 2 4 6 8 10 12 14
Lip
oly
tic a
ctivity
(IU
/mL)
Time (days)
b)
Figure 16: Extracellular lipolytic activity profile determined in M. aphidis cultured for the conditions: a)
[80g/l SBO] (Dashed line with ▲) and [40glu,0:40glu,4] (Line with ●); b) [40glu,0:40sbo,4] (Line with ) and [40sbo,0:40glu,4] (Line with ▲).
40
By analysing the hydrolytic activity profiles observed for the condition that only used glucose
([40glu,0:40glu,4) in both species, it is possible to observe an increase of activity through the
days, with more relevance in M. antarcticus, which have reached 3.8 IU/mL (figure 17 a), 6 times
higher the lipolytic activity observed with M. aphidis (0.56 IU/mL, figure 16a). Although, using 80
g/l of SBO, the values of extracellular lipolytic activity decreased when compared to the condition
of glucose as a sole carbon source, the same behaviour was reported by Arévalo80 for M.
antarcticus.
Although soybean oil is a substrate for lipases, yeast cells seem to optimize lipase production
to cleave soybean oil presented in the culture medium, while lipase production was favoured
when glucose is used, which led to higher extracellular lipolytic activities (figure 17 and 16a)
By analysing figure 16b (M. aphidis-based cultivation) and figure 17b (M. antarcticus-based
cultivation), when the fermentations starts with SBO, the activity is lower than when starts with
glucose, obviously, at day 4, the condition where glucose is added, ([40glu,0:40sbo,4]), lipase
activity increased trough the days, however, when soybean oil is added at day 4, the activity
decreased. One more time, it seems that the cell only produces the minimum lipases to cleave
the oils presented in the culture medium.
These results support the fermentation process that starts with glucose as sole carbon source,
which is a process that is favourable for cellular growth and, importantly, the production of
extracellular lipolytic activity, allowing a fast oil uptake when added after cellular growth. This can
explain the results of MEL production obtained for the condition [40glu,0:40sbo,4] in comparison
with MEL production in [40sbo,0:40glu,4] condition (see figure 14a and e).
Morita et al12 have also determined lipase activity for both strains: for M. antarcticus T-34 using
40 g/l and 120 g/l, lipase activity has reached a maximum of 9 and 5 IU/ml, respectively. These
values are contradictory to what was observed in these results, since the highest activity of lipases
was obtained after feeding the culture medium with glucose. For M. aphidis ATCC 32657 in the
presence of 40 g/l of SBO lipase activity reached a maximum of 6 IU/mL, although in the presence
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
0 2 4 6 8 10 12 14
Lip
oly
tic a
ctivity
(IU
/mL)
Time (days)
a)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0 2 4 6 8 10 12 14
Lip
oly
tic a
ctivity
(IU
/mL)
Time (days)
b)
Figure 17: Extracellular lipolytic activity profile determined in M. antarcticus cultured in the conditions: a) [80g/l SBO] (Dashed line with ▲) and [40glu,0:40glu,4] (Line with ●); b)
[40glu,0:40sbo,4] (Line with ) and [40sbo,0:40glu,4] (Line with ▲).
41
of glucose there was no extracellular lipolytic activity. These differences can be explained by the
differences in strain.
4.2 Production of MEL by mixed carbon source strategy utilization in bioreactors
This section tries to explore the production of MEL comparing both strains in a bioprocess
development perspective, making use of a bioreactor in: a scale-up perspective; more controlled
environment. Furthermore, MEL production in bioreactor is a subject present in few reports,
mainly using large concentrations of SBO, which are still far from a sustainable bioprocess.
Following the results observed in shake-flaks, one of the conditions able to achieve a
considerable MEL titre and low residual fatty acids is the one that starts with 40 g/l of glucose
and, at day 4 a pulse feeding of 20 g/l of SBO ([40glu,0:20sbo,4]) is performed. Therefore, this
was the condition chosen to work in bioreactors, although, due to the formation of foam, after the
1st day of fermentation in bioreactors, to control the foam, the pulse of 20 g/l of SBO was added.
Two situations of total 20 g/l of oil were studied: 1 feed of 20 g/l of SBO after the 1st day of
fermentation and several pulses of 2 g/l of SBO everyday (until day 11) totalizing 20 g/l of oil
added.
• Moesziomyces aphidis
The results obtained for one feed of 20 g/l of SBO are represented in figure 18. Considering
MEL production (figure 18a) it is possible to observe that until day 3 of fermentation, MEL
increased while soybean oil decreased. Although, after day four of fermentation, small red
aggregates, as observed in chapter 4.1.2, appeared in the cultivation (figure 19), reflecting the
production of MEL. These aggregates increased until day seven, and after day nine of
fermentation the aggregates disappeared in the culture medium, increasing MEL concentration
determined from the cultivation samples until 12.8 g/l.
0
5
10
15
20
25
0 1 2 3 4 5 6 7 8 9 10 11 12
ME
L,
FA
(g/L
)
Time (days)
a)
0
5
10
15
20
25
30
35
40
45
0 1 2 3 4 5 6 7 8 9 10 11 12
Dry
-weig
ht, G
lucose (
g/L
)
Time (days)
b)
Figure 18: Production of MEL in bioreactors with M. aphidis, starting with 40g/l of glucose and 1 feed of
20g/l of SBO after the first day. a) MEL production (Line with ●) and FA consumption (Dashed line with ▲);
b) Glucose consumptions (Dashed line with ▲) and biomass (Grey Line ■). Red figures represent the days that appeared aggregates.
42
Figure 19: Evolution of the red aggregates in M. aphidis cultivation in bioreactor using 40 g/l of glucose and after 1 day, 20 g/l of soybean oil were fed: a) day 4; b) day 5; c) day 6 and d) day 9 of fermentation
Feeding strategy in previous section was performed at day 4 of fermentation. Here SBO
addition was performed at the day 1, and to compare with shake-flask in the same condition an
experiment was performed in shake flask following the same feeding strategy and the results are
represented in appendix (appendix 3).
So, it is possible to observe higher MEL titres in bioreactor (12.8 g/l) if compared to the shake
flasks (8.1 g/l). Considering the fatty acids consumptions, 4 g/l remained in the medium, and in
shake flask was 2.2 g/l. It seems that with a high feed of SBO, M. aphidis would be able to produce
more MEL and maintain a low accumulation of FA.
The next experience, instead of add 20 g/l of SBO after one day of fermentation, small pulses
of 2 g/l were added each day, for 10 days. By analysing the results (figure 20) it is possible to
observe that MEL production was 5.8 g/l, lower than results obtained for one feed of 20g/l of SBO,
and a high amount of fatty acids remained in the culture medium (9.1 g/l). So, it seems that small
feeds of SBO are rapidly consumed by the cell and it is enough to produce MEL, accumulating
fatty acids trough the days.
a) b) c) d)
0
5
10
15
20
0 2 4 6 8 10 12
ME
L,
FA
(g
/L)
Time (days)
a)
0
5
10
15
20
25
30
35
40
45
0 1 2 3 4 5 6 7 8 9 10 11 12
Dry
-weig
ht, G
lucose (
g/L
)
Time (days)
b)
Figure 20: Production of MEL in bioreactor with M. aphidis, adding 2g/l of SBO for 10 days: a) MEL
production (Blue line ●) and FA consumption (Black line ▲); b) Glucose consumptions (Dashed line with
▲) and biomass (Grey line ■).
43
By analysing table 9, it is possible to observe that the best yield is for condition involving one
feed of 20 g/l of SBO in bioreactor.
Rau et al101 have tested two strains in bioreactor: M. aphidis DSM 70725 and M. aphidis DSM
143930. In the first strain two studies were performed: starting only with soybean oil (67 g/l) and
starting with soybean oil (67 g/l) and more 37 g/l added in first days of fermentation, and the yields
were 0.53 and 0.67 gMEL/gsubstrate, respectively. The yield obtained in this section is lower, although,
it is a bioprocess that used lower amounts of SBO, 20 g/l, avoiding high accumulation of residual
FA in the cultivation broth.
Using the strain M. aphidis DSM 14390, the authors obtained one of the best yields observed
in the literature, 0.92 gMEL/gsubstrate. However, it is important to notice that beyond the use of
soybean oil and glucose in high concentrations, yeast extract (16 g/l) and nitrate (14 g/l) were
also supplied after one day of fermentation. There are some limitations in this study, since no
information on how much SBO is used by the yeast cell and how to control the foam is available.
Kim et al79 have tested three types of fermentation Candida sp. SY16: batch fermentation, fed-
batch fermentation and foam-stat fed-batch, where the yields were 0.48, 0.45 and 0.50
gMEL/gsubstrate, respectively. The yields are not far from the yield obtained in this experience, with
60 g/l of carbon of source.
Table 9: Summary of MEL obtained at day 14th of fermentation, yield (product/substrate), yield in mol,
purity factor and productivity for conditions 1 feed of 20g/l of SBO in bioreactor and in shake flask, and for the condition with feeds of 2g/l
Strain Condition MEL a)
(g/l)
FA (g/l)
a)
Yield b) (p/s)
Yield c) (molP/molS)
Purity d)
Factor
Productivity e) (g/l.h)
M.aphidis
Feed of 20g/l 12.83 4.54 0.214 0.080 0.739 0.038
Shake Flask 8.11 2.20 0.135 0.051 0.794 0.024
Feed of 2g/l 5.77 9.19 0.096 0.037 0.385 0.017
a) MEL and FA produced at day 14th of fermentation
b) Yield (Titre of MEL produced/ Concentration of carbon source added)
c) Yield (mol of carbon in MEL produced/ total mol of carbon added)
d) Purity factor (MEL produced/ (MEL produced + concentration of fatty acids))
• Moesziomyces antarcticus
Adamczak et al109 have studied two types of feeds of SBO, with two stage feeding in 80 g
portion of SBO and three stage feeding in 60 g portion of SBO, the titre achieved was 28 g/l and
10 g/l, respectively. Although, when used only soybean oil (80g/l), the titre achieved was 45 g/l,
which seems that several feeds of SBO can inhibit MEL production.
In that way, considering the results obtained by Adamczak et al109 for M. antarcticus, this was
not performed a bioreactor with small pulses of 2 g/l of SBO, as performed for M. aphidis.
Therefore, it was only used the condition starting with 40 g/l of glucose and after 1 day, one feed
of 20 g/l.
This experience was performed in duplicate, however, one of the replicates in the first 8 hours
was set with the minimum agitation by technical errors in the bioreactor, and so, the growth of the
strain was delayed comparing with the other bioreactor. Since all the parameters between the two
44
bioreactors are different (glucose consumption, biomass, MEL production and FA) the results are
represented in figure 21 (bioreactor that occurred without errors) and figure 22 (bioreactor where
the agitation was set with the minimum agitation for 8 hours).
By analysing the results of figure 21, it is possible to observe, that until day five of fermentation
MEL production increases rapidly, until 10.5 g/l with a productivity of 0.087 g/l/h. Although, after
day five of fermentation, MEL starts to decrease until values of 0.41 g/l. Even the biomass
decreased through the days until 1.5 g/l, which can be explained by the lack of substrate, meaning
that, this specie is so optimized in consuming oil, that after day 7 of fermentation there was no
more oil in the medium, just small fatty acids produced by the yeast cell.
By analysing figure 22, where the bioreactor was set with a minimum agitation for 8 hours, it
is possible to observe that the tendency is similar, with the difference that the maximum of MEL
production is reached at day 4 of fermentation (8.5 g/l) and then, MEL production and biomass
have decreased through the days, as observed in figure 23. Abadias et al110 tested several
parameters (such as the agitation) to observe the effect in the growth of Candida Sake CPA-1,
0
5
10
15
20
25
0
5
10
15
20
25
30
35
40
0 1 2 3 4 5 6 7 8 9 10 11 12
ME
L,
Fatty a
cid
s (
g/l)
Dry
weig
ht, G
lucose (
g/l)
Time (days)
0
5
10
15
20
25
0
5
10
15
20
25
30
35
40
0 1 2 3 4 5 6 7 8 9 10 11 12
ME
L,
Fatty a
cid
s (
g/l)
Dry
weig
ht
, G
lucose (
g/l)
Time (days)
Figure 21: Production of MEL in bioreactor with M. antarcticus, adding one feed
of 20g/l of SBO: a) MEL production (Blue line ■) and FA consumption (Black
line▲); b) Glucose consumptions (Dashed line with ●) and biomass (Grey line ■).
Figure 22: Production of MEL in bioreactor with M. antarcticus, adding one feed
of 20g/l of SBO: a) MEL production (Blue line ■) and FA consumption (Black
line▲); b) Glucose consumptions (Dashed line with ●) and biomass (Grey Line■).
45
where they have seen that agitation speed influences the oxygen dissolved, as well the growth of
the specie. This explains why the biomass have just reached 17 g/l instead of 23 g/l.
Since there was no MEL in the end, it was constructed a table (table 10), comparing the values
for MEL at day five of fermentation in bioreactor and day ten, in shake flaks (appendix 2).
Table 10: Resuming of the MEL obtained at day 10th of fermentation for shake flask, and day 5th of fermentation for the bioreactor. Also, the yield (product/substrate), yield in mol, and productivity.
a) MEL and FA produced at day 5th of fermentation in bioreactor and day 10th of fermentation in shake flask
b) Yield (Titre of MEL produced/ Concentration of carbon source added)
c) Yield (mol of carbon in MEL produced/ total mol of carbon added)
d) Purity factor (MEL produced/ MEL produced + concentration of fatty acids)
By comparing the values obtained for shake flaks and bioreactor, it is interesting to observe
that at day 5 of bioreactor, MEL production is close to the production in shake flask day 10 of
fermentation. These results are promissing and show that the additions of oil should be made at
day 3 or 4 of fermentation in bioreactor and the concentration of SBO supplied can be higher.
Comparing both species in bioreactor (one feed of 20 g/l), at day 5 of fermentation, the
concentration of MEL was 10.5 g/l in M. antarcticus, and in M. aphidis the concentration was 3.4
g/l of MEL, and it reached 10.0 g/l of MEL only at day ten of fermentation. As discussed in all the
chapters, M. antarcticus is more able to produce MEL than M. aphidis, and the productivity in
bioreactor is much higher for M. antarcticus. Even though M. aphidis should not be discarded due
to the ability of M. antarcticus at forming biofilms, as represented in figure 25, which could be
bring some serious with the fermentation process.
Strain Condition MEL a)
(g/l) FA a)
(g/l)
Yield b) (p/s)
Yield c) (p/s) mol
Productivity e) (g/l.h)
M. antarcticus
Bioreactor 10.54 10.49 0.176 0.066 0.031
Shake flask
12.54 2.42 0.209 0.079 0.037
Figure 23: Image of the biofilm formed in bioreactor
with M. antarcticus
46
4.2.1 Lipolytic activity in bioreactors
Considering the results obtained for M. aphidis and M. antarcticus, extracellular lipolytic activity
was also measured, to understand if the enzymatic activity follows the same trend as observed
in chapter 4.1.6.
By analysing figure 24, it is possible to observe that for the condition of one feed of 20 g/l of
SBO, the activity starts to increase, due to the presence of glucose, as observed in figure 20.
After day 6 of fermentation, the activity stabilized and stop increasing. For the condition where
small pulses of 2 g/l were added every day, it also increased until day 6 of fermentation, and after
that, the activity increased to values of 1.28 U/mL.
Comparing both conditions, after day 6, the lipase activity is much higher for small pulses of 2
g/l of SBO than for one single feed of 20 g/l of SBO. As discussed in chapter 4.1.6, it seems that
in the presence of oil, the cells just produce the minimum of lipases to cleave the triglycerides
presented in oil. This could justify why small pulses of 2 g/l of SBO exhibit high activity of lipases,
since it is cleaved fast due to the small concentration of oil, and then, it seems that the cell
produces more lipases to find oleaginous compounds.
By observing the results obtained for M. antarcticus (Figure 25), enzymatic activity increased
until day 3, due to the presence of glucose, as observed in figure 21a. After day six of
fermentation, the day that MEL starts to decrease (Figure 21), enzymatic activity starts to
increase, reaching values of 6.2 IU/mL. Again, it seems that when SBO is present, the activity is
lower, and after SBO is consumed, the cell produces more lipases as described to M. aphidis.
If this theory is confirmed and also observed by Arévalo80, enzymatic activity could be a way
of indirectly measuring the presence of oil in the fermentation broth.
0.0
0.4
0.8
1.2
1.6
2.0
0 2 4 6 8 10 12
Lip
oly
tic a
ctivity
(U/m
L)
Time (days)
Figure 24: Extracellular lipolytic activity profile determined in M. aphidis cultured on 40 g/l of glucose and: 1 pulse feed of 20 g/l of SBO (Black line
■) and several pulse feeds of 2 g/l (Dashed line with ●).
47
4.3 Producing MEL using waste frying oil (WFO)
To industrialize the production of a given biological product, one of the objective is to use
renewable substrates, to reduce the cost of fermentation. In USA, 100 billion litter of used oils are
being produced per week by all the restaurants 106, and so due to the high volume of production
of these wastes, they could be used to produce MEL, instead of soybean oil, since the land
needed for this crop is increasing, also, the water consumption needed, as described by Schmidt
et al107.
In that way, this chapter tries to observe if the production of MEL is affected, when soybean
oil is replaced by waste frying oil. Although, at a first trial, this experience was only performed in
shake-flask.
Hence, one of the best conditions achieved in producing MEL and low accumulation of FA was
used, replacing WFO for SBO. Therefore, all the fermentations started with 40g/l of glucose and
at the 4th day,20 g/l of WFO was supplied to the medium ([40glu,0:20wfo,4]).
By analysing the values obtained with WFO for M.aphidis (figure 26), it is possible to see that
the production of MEL reached 10 g/l of MEL, and FA are almost completely consumed.
0
2
4
6
8
10
0 2 4 6 8 10 12
Lip
oly
tic a
ctivity
(U/m
L)
Time (days)
0
5
10
15
20
25
0
5
10
15
20
25
30
35
40
45
0 2 4 6 8 10 12 14 ME
L (
g/l),
Fatty a
cid
s (
g/l)
Dry
weig
ht
(g/l),
Glu
cose
(g/l)
Time (days)
Figure 26: Production of MEL (Dashed line with ▲), consumption of FA (Line with
▲), formation of biomass (■) and glucose consumption (Dashed line with ●) by M.
aphidis for conditions [40glu,0:20wfo,4].
Figure 25: Extracelular lipolytic activity profile determined in M. antarcticus cultured on 40 g/l of glucose
and 1 pulse feed of 20 g/l of.
48
Figure 27: Production of MEL (Dashed line with ▲), consumption of FA (Dashed line with ▲),
formation of biomass (Line with ■) and glucose consumption (Dashed line with ●) by M. antarcticus for
conditions [40glu,0:20wfo,4].
Considering the results for M. antarcticus (figure 27) it is possible to observe MEL titre of 12.1
g/l, not far from the results obtained when SBO is used (14.8 g/l). The consumption of FA was
very similar in both cases. Obviously, by observing table 9 it is possible to see that yields for SBO
are higher than the yields for WFO.
In table 11 it is possible to compare the yields obtained for SBO and WFO for both species,
where it is possible to see that the production of MEL almost reaches the amount obtained using
SBO, even though, neither of the parameters have improved using WFO.
Table 11: MEL and FA production, yields, purity factor and productivity after 14 days of M. aphidis and M. antarcticus cultured on 40 g/l of glucose and pulse of WFO at day 4.
a) MEL and FA produced at day 14th of fermentation
b) Yield (Titre of MEL produced/ Concentration of carbon source added)
c) Yield (mol of carbon in MEL produced/ total mol of carbon added)
d) Purity factor (MEL produced/ (MEL produced + concentration of fatty acids))
For both species, the addition of WFO did not lead to higher values than SBO, and this could
be justified by the fact WFO is more oxidized than SBO. Oxidation of polyunsaturated lipids is the
main reaction of lipids degradation, involving the generation of free radicals, and could be harmful
for the yeast culture. This autoxidation can be stimulated by light, ionizing radiation even by the
enzyme lipoxygenase (EC 1.13.11.-) 108. In this way, Arévalo 80 have characterized several oils,
including WFO and SBO, and one of the parameters analysed was acid value, which indicates
the amount of free fatty acids on oils. Therefore, the values obtained by Arévalo, for WFO was
Strain Condition MEL a)
(g/l) FAa)
(g/l) Yield b)
(p/s)
Yield c) (p/s) mol
Purity d)
Factor Productivity
e) (g/l.h)
M. aphidis WFO 10.01 1.51 0.167 0.042 0.869 0.030
SBO 11.59 3.00 0.193 0.073 0.794 0.034
M. antarcticus
WFO 12.09 1.43 0.202 0.048 0.893 0.036
SBO 14.84 1.47 0.247 0.0943 0.910 0.044
0
5
10
15
20
25
0
5
10
15
20
25
30
35
40
45
0 2 4 6 8 10 12 14
ME
L (
g/L
), F
atty a
cid
s (
g/L
)
Dry
weig
ht, G
lucose (
g/L
)
Time (days)
49
4.67 mgKOH/g and for SBO was 1.29 mgKOH/g. Other parameters were also determined in that
study, such as saponification value, which are presented in appendix (appendix 1).
And so, knowing that MEL production occurs by partial β-oxidation of fatty acids, known as
chain shortening pathway 62, and if WFO have an acid value higher than SBO, this means that
there are less fatty acids to incorporate into the formation of MEL, explaining why SBO have
produced more MEL.
In the literature Arévalo, using 70 g/l of WFO, obtained 8.3 g/l of MEL, a difference of 53.8 %
when compared to the production of MEL using the same amount of SBO (18 g/l). However, in
the present section, the values obtained were very close to the values obtained for SBO, with a
difference of 13.6% for both species. This means that glucose plays an important role at preparing
the cell, as discussed earlier and WFO should be considered for further studies.
4.4 Downstream processing by nanofiltration technology
This section tries to elucidate if it possible to separate MEL from fatty acids, using
nanotechnology (nano-membranes). In this work, tested two membranes were tested: 22% of PBI
(ranging from 540 to 580 Da) and with larger MWCO membrane with 17% of PBI.
For all these studies, MEL and FA extracted from the bioreactor performed with M. aphidis
supplying one feed of 20 g/l of SBO, were used.
4.4.1 Testing the membrane with 22% of PBI solution
Fatty acids are composed by: monoglycerides (MAG), diglycerides (DAG), both with a size
inferior than 580 Da and triglycerides (TAG) larger than 580 Da. In theory, using this technology,
it will be possible to separate MAG and DAG from MEL.
With this membrane, three types of solvents at different pressures were tested: Ethyl acetate
(10, 20 e 30 bar), Isopropanol (30 bar), and MTBE (30 bar). Pre-conditioning membrane and
solvent treatment have influence in rejection and fluxes. In that way, Razali et al 111 have
performed several testes with different organic solvents and the same membrane used in this
thesis (22% of PBI). The authors have found that to obtain a rejection coefficient of 100% without
compromising the flux, the best organic solvents to pre-conditioning the membrane are acetone
and Ethyl acetate. In that way, in each filtration performed, ethyl acetate was always used to pre-
conditioning the membrane.
50
By analysing figure 28, for ethyl acetate, the flux increases as the pressure increases.
Although, when the pressure is equal, and the solvent is changed it will depend on the polarity of
the membrane and the organic solvent. As described by Razali et al 110, the permeability of the
membrane increases as the polarity of organic solvents increases. Therefore, since ethyl acetate
is the solvent more polar when compared with isopropanol and MTBE, the flux is much higher.
For all the conditions, the rejection coefficient (Rc) for MEL, monoglycerides and triglycerides
were calculated using equation 2.
𝑅𝑐 (%) = (1 −𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑖𝑛 𝑝𝑒𝑟𝑚𝑒𝑎𝑡𝑒
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑖𝑛 𝑟𝑒𝑡𝑒𝑛𝑡𝑎𝑡𝑒) ∗ 100 (Equation 2)
Considering the values of rejection coefficient of MEL for all the conditions (figure 29), most of
the conditions have the same rejection, around 98%.
Then analysing the results obtained of these filtrations for MAG (monoglycerides), figure 31a,
it is possible to observe that the all the conditions present similarly rejection coefficients, however,
MTBE and isopropanol gave the best result. For condition 20 bar using ethyl acetate, the rejection
coefficient was around 60%, better than the 30 bar, using ethyl acetate.
0
2
4
6
8
10
12
10bar Eth_Ac 20 bar Eth_Ac 30 bar Eth_Ac MTBE Isopropanol
Flu
x (L
/h/m
2)
Figure 28: Flux for each condition: 10 bar ethyl acetate (black bar), 20 bar ethyl acetate (blue bar), 30 bar Ethyl acetate (grey bar); MTBE (orange bar) and isopropanol (green bar)
0%
20%
40%
60%
80%
100%
10 bar Eth_Ac 20 bar Eth_Ac 30 bar Eth_Ac MTBE Isopropanol
Rej
ecti
on
Co
effi
cien
t (%
)
Figure 29: Rejection coefficient of MEL for each condition: 10 bar ethyl acetate (black bar), 20 bar ethyl acetate (blue bar), 30 bar Ethyl acetate (grey bar); MTBE (orange bar)
and isopropanol (green bar)
51
Considering the results of rejection coefficient for triglycerides it is possible to observe (figure
30b) that for all the conditions using the solvent ethyl acetate, the rejection is 100%, which makes
sense, since the MWCO range from 540 to 580 Da and triglycerides have a molecular weight of
885. Although, the rejections for MTBE and isopropanol are 48% and 75% with an error significant
(in both graphs for MAG and TAG). This can be explained by the weak interaction with the
membrane, causing a low flux (figure 28), and so, there is more time for “leaks” and some of the
components will be able to cross around the membrane.
Considering the initial mass of MEL (55 mg) and FA (12 mg) that is presented in each solution,
the losses of both compounds after the filtration occurred, were calculated. Therefore, by
analysing the figure 31, it is possible to observe that the conditions that exhibit more losses of
MEL (figure 31a) and FA (figure 31b) were MTBE and isopropanol due to the possible “leaks”
during the filtrations, as earlier explained.
0%
20%
40%
60%
80%
100%
Rej
ecti
on
Co
effi
cien
t (%
)
Conditions
a)
0%
20%
40%
60%
80%
100%
120%
Rej
ecti
on
co
effi
cien
t (%
)
Conditions
b)
Figure 30: Rejection coefficient for monoglycerides (a) and triglycerides (b) for each condition: 10 bar ethyl acetate (black bar), 20 bar ethyl acetate (blue bar), 30 bar Ethyl acetate
(grey bar); MTBE (orange bar) and isopropanol (green bar)
Figure 31: Percentage of masses and losses for MEL (a) and FA (b), for the conditions 10 bar ethyl acetate (black bar), 20 bar ethyl acetate (blue bar), 30 bar Ethyl acetate (grey bar); MTBE (orange bar) and isopropanol (green bar). Orange bars represent the losses of the compounds.
0
10
20
30
40
50
60
70
80
90
100
Mas
s (%
)
Conditions
a)
0
10
20
30
40
50
60
70
80
90
100
Mas
s (%
)
Conditions
b)
52
Considering all the results obtained with this membrane, the best result was the use of ethyl
acetate as organic solvent with 20 bar of pressure, since the rejection coefficient for MEL and FA
is 98% and 60%, respectively, and is one of the conditions that have less losses of MEL and FA.
Although, in the presence of triglycerides, there is no possibility of separate MEL from TAG, and,
in that way two approaches were performed: enzymatic activity, using CAL-B (lipase) to observe
if the residual triglycerides are cleaved and using a larger membrane (17% of PBI) and try to
separate TAG and MAG from MEL with a better rejection coefficient.
Gueiros111, in her master thesis, have used nanofiltration technology to separate MEL from
long chain methyl esters, using a commercial membrane (starmem240 (400 Da)). The rejection
coefficient for MEL, MAG (monoolein – 1-monooleoyl-rac-glycerol) and TAG (Trioelin) was 94%,
34% and 95.2%, respectively. Comparing the values obtained to this study, considering MAG are
lower than the value obtained (60%). Although, this difference can be explained, to the fact, n-
hexane is used to prepare solutions of MEL, MAG and TAG, and the membrane used in that
study, is commercial. Furthermore, the solutions used are more “clean” just pure compounds,
instead of the solutions used in this study, which have MEL, MAG, TAG and small fatty acids.
4.4.2 Enzymatic reaction to breakdown triglycerides
To perform this reaction, a solution of 2 g/l in a total volume of 5 ml of phosphate buffer at pH
7 was prepared, and then 0.2 mg of the enzyme (CAL-B) were add to the solution and the reaction
was let in incubator for seven days at 37ºC.
Observing figure 32, it is possible to observe the spectre of the aggregates of MEL and FA,
where it is possible to observe residual triglycerides (red rectangle) and monoglycerides (yellow
rectangle). The others peak seems to be a type of diglyceride.
Then after the enzymatic reaction occurred, all the solution was extracted to observe the profile
of glycerides and, to observe if MEL structure was affected. By analysing the spectre after the
reaction occurred (figure 33), it is possible to observe that the triglycerides were consumed with
Figure 32: HPLC spectre of the aggregates of MEL and FA from the bioreactor with M. aphidis and used to perform filtrations. Blue rectangle
correspond to MAG and red rectangle corresponds to TAG.
53
a conversion of 100%, and the area of MAG increased from 10 million to 40 million. If in the end
of the fermentation, the culture medium still has TAG, it means that it is possible to add CAL-B,
or even the supernatant enriched in lipases, adding to the bioreactor and let the enzymatic
reaction occurs for some days, allowing a better separation of MEL from FA (principally
monoglycerides).
Knowing that triglycerides can be cleaved by the action of CAL-B and by assuming that the
final extraction of the bioreactor was only constituted by free fatty acids, MAG and MEL, some
calculations were performed in order to determine how diavolumes are required to obtained MEL
almost pure, as described in table 14. For these calculations, it was assumed the best conditions
achieved in section 4.3.1, which was ethyl acetate at a pressure of 20 bar and rejection coefficient
for MEL and FA of 98% and 60% respectively.
Therefore, as represented in equation 3, was calculated the concentration in the retentate (cR)
for FA and MEL, knowing that the concentration in the feed (cF) was the same concentration of
MEL and FA in the aggregates used to filtrate (14.85 g/l to MEL and 3.85 g/l to FA), considering
the rejection coefficient (Rc) for each diavolume (D)
𝑐𝑅 = 𝑐𝐹 ∗ 𝑒(−𝐷∗(1−𝑅𝑐)) (equation 3)
Figure 33: HPLC spectre, after the enzymatic reaction have occurred. Blue rectangle corresponds to MAG, and red rectangle
corresponds to the zone, where TAG should appear.
54
Table 12: Theoretical calculation of concentration in retentate (cR) for FA and MEL, % of FA in the feed and MEL purity (%), assuming a rejection coefficient for MEL and FA of 98% and 60%, respectively and a
concentration of MEL (14.85 g/l) and FA (3.85 g/l)
FA MEL
Diavolumes cR
(g/l) %FA in the
feed cR
(g/l) MEL purity %
% of MEL lost
1 2.58 67.03 14.55 85.19 2.04
2 1.73 44.93 14.26 89.56 3.98
3 1.16 30.12 13.98 92.75 5.89
4 0.78 20.19 13.70 95.02 7.75
5 0.52 13.53 13.43 96.61 9.58
6 0.35 9.07 13.16 97.70 11.37
7 0.23 6.08 12.90 98.45 13.12
8 0.16 4.08 12.65 98.95 14.84
By analysing the values of these table, it seems that using 5 diavolumes it is possible to reach
96.61% of purity. In practically, performing diavolumes the rejection coefficient tends to decrease,
since the filtration mode will operate in cross flow.
4.4.3 Testing the membrane with 17% of PBI
The next objective was to use a larger MWCO membrane, observing the behaviour of MEL,
monoglycerides and triglycerides. Since this MWCO is higher than the molecular weight of MEL,
to avoid the passage of MEL to the permeate, it was attempt the creation of micelles.
For that, 50 mL of water and 50 mL of ethyl acetate were mixed and warm up to help the
solubilization of water in ethyl acetate and vice-versa. After that, organic phase (OP) was
separated from the aqueous phase (AP), and both were used to solubilize 2 g/l of MEL and FA.
Both solutions were sonicated for 10 minutes, in order to give energy to the system, allowing the
formation of micelles.
Analysing figure 34, it is possible to observe that the flux for organic phase is much higher
than the flux obtained for the aqueous phase. This difference in flux can be explained by the low
interaction of the water with the membrane, as explained before.
0
5
10
15
20
25
30
35
40
45
Organic phase Aqueous phase
Flu
x (L
/h/m
2)
Figure 34: Flux for each condition: organic phase (black bar) and aqueous phase (blue bar)
55
In figure 35 the rejection coefficient for MEL in both conditions are represented. The rejection
for organic phase is 16%, which indicates that MEL did not form the micelles, crossing the
membrane as expected. For aqueous phase, the rejection coefficient for MEL is 63%, which
suggests the formation of micelles, but not all the MEL have formed micelles and some has
passed the membrane.
Then, by analysing the rejection coefficients for MAG (figure 37a) and TAG (figure 37b), it is
possible to observe that in the organic condition, the values for MAG (6%) and TAG (18.1%) are
very low, as expected, since this membrane used have a high MWCO. For the aqueous phase,
the rejection coefficients are a bit higher than the values for organic phase, although, the values
are good, 12.9% for MAG and 44.2% for TAG, since the rejection coefficient for MEL is 63%.
Imura et al 112 have reported the self-assembling properties of MEL-A and MEL-B, where they
found that both types of MEL does not self-assemble in a micelle form, but into large unilamellar
vesicles (LUV),as with a CAC (critical aggregates concentrion) of 4.0*10−6 M for MEL-A and
6.0*10−6 M. Although the authors found that the structure of MEL-A above CACII (1.0*10-3 M)
0%
20%
40%
60%
80%
100%
Organic phase Aqueous phase
Rej
ecti
on
co
effi
cien
t (%
)
a)
0%
20%
40%
60%
80%
100%
Organic phase Aqueous phase
Rej
ecti
on
co
effi
cien
t (%
)
b)
Figure 36: Rejection coefficient for organic phase and aqueous phase: a) monoglycerides and b) triglycerides
0
20
40
60
80
100
Organic phase Aqueous phase
Rej
ecti
on
co
effi
cien
t (%
)
Figure 35: Rejection of MEL for both phases. Organic phase (black bar) and aqueous phase (blue bar)
56
changed completely, to a typical morphology of a sponge structure. The complete difference from
the two structures created by MEL-A have provided two CAC for MEL-A.
This results obtained by Imura et al 112, explain why the rejection coefficient for MEL, in
aqueous phase have increased. However, more studies are required, mainly to obtain more
micelles, increasing rejection coefficient for MEL and allowing a better separation of MEL from
MAG and TAG.
57
Chapter 5 - Conclusions
The two main objectives were: the elucidation of a medium capable of producing high titres of
MEL, ending the fermentation with the minimum fatty acids present in the medium, and improving
the separation of MEL and FA using nanofiltration technology.
This study showed that the fermentation should start with glucose (40 g/l) and, only after four
days of fermentation, SBO or other oil should be added. In that case, two types of feed were
tested (40 g/l and 20 g/l of SBO). The feed of 40 g/l have led to the best titre of MEL observed in
this work, 24 g/l for M. antarcticus and 18 g/l for M. aphidis, with a yield of 0.309 and 0.222
gMEL/gsubstrate, respectively, with the amount of FA being considerable high. The feed of 20 g/l have
led to a yield of 0.193 gMEL/gsubstrate for M. aphidis and 0.247 gMEL/gsubstrate for M. antarcticus, ending
the fermentation with almost no residual fatty acids in the medium.
It was also tested the production of MEL starting with glucose (40 g/l) and a feed of WFO in
day four of fermentation. The yields obtained were 0.167 gMEL/gsubstrate for M. aphidis and 0.202
gMEL/gsubstrate for M. antarcticus. In this case, the difference between SBO and WFO was only 13%
for both species, indicating that WFO should be used in future works.
Since the fatty acids in the end of the fermentation is a problem, it was tested the effect of rich
compounds in nitrogen, such as peptone, yeast extract and CSL. These compounds have shown
the ability to produce high concentration of biomass, where peptone have produced just 1 g/l of
MEL in both strains. In the case of yeast extract and CSL the behaviour was different, since it
produced considerable titres of MEL, but did not improve MEL production comparing to the
control, and so, more studies are required to find the optimum concentration of these compounds.
In bioreactor, starting with glucose and a fed of SBO in day 1 day of fermentation, with M.
aphidis, was obtained titre of 12.8 g/l with a yield of 0.214 gMEL/gsubstrate. For M. antarcticus it was
observed an increase production of MEL until day five (10.5 g/l), and then MEL decreased, due
to the totally consumption of SBO. These results are promising, since it shows that it is possible
to increase the concentration of SBO supplied to the medium and maintain a low accumulation of
fatty acids in the cultivation.
Nanofiltration technology was also performed to improve the separation of MEL from fatty
acids, and two membranes were tested with 17% and 22% of PBI. The best results for the
membrane at 22% was when using ethyl acetate to filtrate, with a pressure of 20 bar, where the
rejection coefficient for MEL, MAG and TAG were: 98%, 60% and 95%, respectively. Although
the presence of triglycerides avoids the separation of MEL from TAG with the membrane with
22% of PBI. In that case two approaches were performed, using an enzymatic reaction to cleave
TAG, which was successful. The other approach was to use a membrane with a larger MWCO
(17 % of PBI), where the creation of micelles was tried, by mixture ethyl acetate and water,
separating organic phase from aqueous phase. The coefficient rejection for the organic phase to
MEL, MAG and TAG were 12%, 6% and 18% respectively. These values were expected since
this is a membrane larger and all the compounds have a molecular weight inferior to the
membrane pore. For the aqueous phase, the values of rejection coefficient for MEL, MAG and
58
TAG, were 63%, 12.88% and 44%, respectively. Since the rejection coefficient is higher than for
the organic phase, it seems that MEL formed large vesicles due to the presence of water.
59
Chapter 6 - Future perspectives
To create a sustainable process, renewable substrates should be used. In this thesis waste
frying oil was used with glucose, but in the future, lignocellulosic residues could replace glucose,
creating a bioprocess with only renewable substrates (glucose replacing lignocellulosic residues
and waste frying oil replacing soybean oil). Cheese whey is one example of a renewable substrate
with interest, due to its constitution. In that way, experiments could be performed, using cheese
whey, instead of glucose.
The metabolism of the formation of MEL should be studied in more detail, studying the
participation of each enzyme involved.
In this thesis was evaluated the effect of CSL in both species, where it was produced
considerable amounts of MEL, however, it seems that there are some component inhibiting the
production of MEL. Considering that CSL is constituted by 40% of proteins, 21% of lactic acid,
16% of nitrogen free extract and several vitamins, maybe the high percentage of lactic acid is
interfering with the metabolism. Therefore, some experiments should be performed using several
concentrations of CSL (1, 2, 5 and 10 g/L) and understand what is the best concentration to be
used in the culture medium
After the consumption of soybean oil in the bioreactor, the pH has the tendency to increase till
values of 8, consequently, the pH should be controlled at 6 and analysing if this control of pH will
affect MEL production. Since 20 g/L of SBO are consumed efficiently in a bioreactor, a high feed
of SBO should be supplied, such as 40 g/L, evaluating MEL production as well the consumption
of fatty acids.
In the nanofiltration technology, if the fermentation ends without triglycerides, using a nano-
membrane (540-580 Da) diavolumes should be used to improve the separation of MEL form FA.
However, commercial membranes, with the same range (540-580 Da) or with a lower MWCO,
should be tested to improve separation of MAG and MEL, decreasing the rejection coefficient for
MAG.
In order to use a larger MACO membrane (17% of PBI), more studies are required, especially
in the formation of micelles.
60
61
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Chapter 8 - Appendix
8.1 Appendix 1
Comparison between soybean oil (SBO) and waste frying oil (WFO). Values retrieved from
Arévalo Thesis80 .
Parameter WFO SBO
Acid Value (mg KOH/g) 4.67 1.29
Moisture and volatile matter content (%
m/m)
0.10 0.07
Insoluble impurity content (% m/m) <0.01 0.01
Saponification value (mg KOH/g) 196 195
Iodine value (g I2/100g) 106 130
Unsaponifiable matter (% m/m) n.a. na
Fatty acid chain - -
C14:0 0.1 0.1
C16:0 4.9 11.5
C16:1 0.1 0.1
C18:0 0.1 3.7
C18:1 62.8 23.5
C18:2 27.9 53.5
C18:3 1.5 6.6
C20:0 0.4 0.4
C22:0 0.8 n.d
C24:0 n.d n.d
Others 1.4 0.6
Saturated 6.3 15.7
Unsaturated 92.3 83.7
There are very parameters described in table, and so:
• acid value corresponds to the amount of free fatty acids in the medium, which is an
indication of how much an oil is degraded
• Saponification value: amount of free fatty acids extracted in 1g of samples
• Iodine value: the amount of instaurations present in fatty acids
71
8.2 Appendix 2
In this section the values of MEL, FA and biomass of the section 4.1.1 are represented. As it is
possible to observe, for the high concentrations of SBO, 80g/l (figure A1) and 60 g/l (figure A2),
MEL production is low compared to the values obtained after the extraction, due to the
appearance of red aggregates in the culture medium.
A1: Production of MEL (dashed line), Fatty acids consumption (black line) and biomass production (grey line) in shake flask for the condition [80g/l SBO] in M. aphidis (a) and M. antarcticus (b)
A2: Production of MEL (dashed line), Fatty acids consumption (black line) and biomass production
(grey line) in shake flask for the condition [60g/l SBO] in M. aphidis (a) and M. antarcticus (b)
A3: Production of MEL (dashed line), Fatty acids consumption (black line) and biomass production
(grey line) in shake flask for the condition [40g/l SBO] in M. aphidis (a) and M. antarcticus (b)
0
10
20
30
40
50
60
70
80
90
0
5
10
15
20
25
30
35
0 2 4 6 8 10 12 14
ME
L,
Fatty a
cid
s g
/L
Gro
wth
(g/L
)
Fermentation (days)
b)
0
10
20
30
40
50
60
70
80
90
0
5
10
15
20
25
30
35
0 2 4 6 8 10 12 14
ME
L,
Fatty a
cid
s g
/L
Gro
wth
(g/L
)
Fermentation (days)
a)
0
10
20
30
40
50
60
70
0
5
10
15
20
25
30
35
0 2 4 6 8 10 12 14
ME
L,
Fatty a
cid
s g
/L
Gro
wth
(g/L
)
Fermentation (days)
a)
0
10
20
30
40
50
60
70
0
5
10
15
20
25
30
35
0 2 4 6 8 10 12 14M
EL,
Fatty a
cid
s g
/L
Gro
wth
(g/L
)
Fermentation (days)
b)
0
5
10
15
20
25
30
35
40
45
0
5
10
15
20
25
30
35
0 2 4 6 8 10 12 14
ME
L,
Fatty a
cid
s g
/L
Gro
wth
(g/L
)
Fermentation (days)
a)
0
5
10
15
20
25
30
35
40
45
0
5
10
15
20
25
30
35
0 2 4 6 8 10 12 14
ME
L,
Fatty a
cid
s g
/L
Gro
wth
(g/L
)
Fermentation (days)
b)
72
A4: Production of MEL (dashed line), Fatty acids consumption (black line) and biomass production
(grey line) in shake flask for the condition [20g/l SBO] in M. aphidis (a) and M. antarcticus (b)
8.3 Appendix 3
In this appendix the results of the condition, used in bioreactor for M. aphidis and M. antarcticus
are represented. This condition started with 40g/l of glucose and at the 1st day of fermentation
20g/l of SBO was added to the culture medium.
It is possible to observe in both species that a feed of SBO at day one of fermentation is too
soon, and after day ten of fermentation MEL production decreases, explaining why MEL
production in M. antarcticus (figure 23) decreases after day five. Surprisingly, M. antarcticus is
more efficient at producing MEL and consuming soybean oil in bioreactor than shake flask.
0
5
10
15
20
25
0
5
10
15
20
25
30
35
40
45
0 2 4 6 8 10 12 14
ME
L (
g/L
), F
atty a
cid
s (
g/L
)
Dry
weig
ht
(g/L
), G
lucose (
g/L
)
Fermentation (days)
M.aphidis
A5: Production of MEL in shake flasks for the conditions [0:20] using M.
aphidis: MEL production (Dashed blue line with ■), FA consumption (Black line
with▲), Glucose consumption (Dashed line with with ●) and biomass formation
(Grey line with ■).
0
5
10
15
20
25
0
5
10
15
20
25
30
35
0 2 4 6 8 10 12 14
ME
L,
Fatty a
cid
s (
g/L
)
Gro
wth
(g/L
)
Fermentation (days)
b)
0
5
10
15
20
25
0
5
10
15
20
25
30
35
0 2 4 6 8 10 12 14
ME
L,
Fatty a
cid
s (
g/L
)
Gro
wth
(g/L
)
Fermentation (days)
a)
73
8.4 Appendix 4
A study was performed to quantify the amount of polyphenols in a hydrolysate X (from
lignocellulosic residues), after and before the filtration (where 6 diavolumes were used),
comparing with a liquid fraction of an hydrolysate created within iBB and filtrated the same way
as hydroysate X. The quantification of polyphenols was based in the method described by M.
Amzad Hossain et al113 and adapted to be used in 96 well plate.
The procedure started by adding 40 µL of the sample, then 40 µL of the folin-ciocalteu reagent,
and after 4 minutes, 200 µL of Na2CO3 was added, and incubated at 40ºC for 30 minutes
Jönsson et al114 shown that the presence of polyphenols had influence in the fermentation
process. Therefore, this study is important for future studies using this hydrolysate instead of
glucose.
0
5
10
15
20
25
0
5
10
15
20
25
30
35
40
45
0 2 4 6 8 10 12 14
ME
L (
g/L
), F
atty a
cid
s (
g/L
)
Dry
weig
ht, G
lucose (
g/L
)
Fermentation (days)
M.antarcticus
A6: Production of MEL in shake flasks for the conditions [0:20] using M.
antarcticus: MEL production (Dashed blue line with ■), FA consumption (Black
line with ▲), glucose consumption (Dashed line with ●) and biomass formation
(Grey line with ■).
A7: Quantification of polyphenol for hydrolysate X (blue bars) and liquid fraction (orange bars), after and before the filtration.
0.00
0.50
1.00
1.50
2.00
2.50
Beforefiltration
After filtration Beforefiltration
After filtration
Hyrolysate X Liquid fraction - iBB
Concentr
ation (
g/L
)
Polyphenol concentration
74
8.5 Appendix 5
Considering the nitrogen sources, a fermentation, without replicate was performed, using the
normal components of the medium and adding 2g/l of corn steep liquor, instead of 10g/l as
described in chapter 3. There are only results for M.aphidis.
These results only show one thing, that more tests are required to find the optimum
concentration of Corn steep liquor to increase MEL production, since at day 10 there are 17.09
g/l, higher than the obtained for M.aphidis, using 10g/l of corn steep liquor.
A8: Production of MEL using 2g/l of CSL. MEL production (Dashed blue line ■), FA
consumption (Black line▲); b) Glucose consumptions (Dashed line with ●) and biomass
(Grey line ■).
0
10
20
30
40
50
0
10
20
30
40
50
0 2 4 6 8 10 12 14
MEL
, FA
(g/
L)
Glu
cose
, Dry
wei
ght
(g/L
)
Fermentation (days)
75