Suspension Bioreactor Strategies for Itaconic Acid Production Using Engineered Microorganisms

Suspension bioreactor strategies for itaconic acid production using engineered microorganisms

Nuno Miguel Sousa Marques

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: Professor Helena Maria Rodrigues Vasconcelos Pinheiro Supervisor: Professor Frederico Castelo Alves Ferreira Member of the commitee: Professor Nuno Gonçalo Pereira Mira

November 2017

Acknowledgments It is acknowledged funding from Portuguese Foundation for Science and Technology (FCT) through iBB-Institute for Bioengineering and Biosciences (FCT reference: UID/BIO/04565/2013 and POL2020 reference 007317, including iBB grant ITACYEAST), project CRUISEPTDC/AAG- TEC/0696/2014 and scholarship SFRH/BPD/108560/2015.

During this journey, I would like to thank to my supervisors for all the collaboration and guidance during this process. Also Prof. Dr. Nuno Mira for all the collaboration and availability to help me. To all my work colleagues that during these months helped me, with special references for André Costa, António Maduro, Carlos Rodrigues, Flávio Ferreira, João Santos, Margarida Silva, Marisa Santos, Ricardo Pereira and Dona Rosa. To my colleagues from Biotechnology and for my brothers of another mother, Carlos Fernandes, Miguel Chapado, Miguel Nascimento (Los Abadia), Diogo Mendonça, João Lopes, João Nunes, Miguel Antunes, Pedro Reis, Silvestre Leite, Tiago Nuno Jorge, and all the others I am not mentioning, thank you for all the moments together. For all my Sport Lisboa e Benfica colleagues that during this time help me doing one of the things I like more. To Bara, Pavla and Martina. To Katka for all our time together and all the things we already did. Learn Portuguese to read the Portuguese paragraph. My Slovak is far away better than your English! Last but not the least, to my family, my brother, my mom, my dad, and Benny, thank you for all.

Agradeço ao financiamento da Portuguese Foundation for Science and Technology (FCT) through iBB-Institute for Bioengineering and Biosciences (FCT reference: UID/BIO/04565/2013 and POL2020 reference 007317, including iBB grant ITACYEAST), project CRUISEPTDC/AAG- TEC/0696/2014 and scholarship SFRH/BPD/108560/2015.

Durante esta aventura, tenho de agradecer aos meus supervisores por toda a colaboração, ajuda e conhecimento ao longo deste processo de aprendizagem. Ao Prof. Dr. Nuno Mira por toda a colaboração e disponibilidade para me ajudar. A todos os meus colegas de laboratório que me ajudaram, alguns tornando-se até amigos, com especial referência a André Costa, António Maduro, Carlos Rodrigues, Flávio Ferreira, João Santos, Margarida Silva, Marisa Santos, Ricardo Pereira e claro, à Dona Rosa. A todos os meus colegas de Biotecnologia e para os meus irmãos de outra mãe, Carlos Fernandes, Miguel Chapado, Miguel Nascimento (Los Abadia), Diogo Mendonça, João Lopes, João Nunes, Miguel Antunes, Pedro Reis, Silvestre Leite, Tiago Nuno Jorge (por todo o meu conhecimento de NBA que te vai fazer rico deves-me (mais) uma imperial). A todos os não mencionados, um obrigado por todos os momentos juntos. A todos os meus colegas do Sport Lisboa e Benfica com quem durante este tempo partilhei muitas horas da minha vida, o meu obrigado. Só vos menciono em português porque o Mota só fala chinês agora malta. Por isso obrigado Diogo, Mota, Marc, Bernardo e Andrés e Nuno Azevedo por termos passado

ii tantas horas juntos que nunca foram desperdiçadas. À Bára, Pavla e Martina. À Katka por tudo o que vivemos juntos. Vê lá se aprendes português, eu já falo eslovaco fluententemente… E claro, aos meus pais, o maior motivo para escrever em português aqui (tenho de vos pagar o curso de inglês?). Ser o primeiro licenciado cá de casa só me mostrou que os cursos valem muito pouco comparado com aquilo que sabemos fazer. E não definem e nunca vão definir quem somos ou quem seremos. Usar o que sabemos fazer no que queremos fazer. E se não sabemos, aprendemos. E ao meu irmão querido, que iniciou agora a sua vida académica, espero que não te percas para o álcool e afins.

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Abstract The present work aims to study the potential conditions for production of itaconic acid (ITAC) using engineered microorganisms (Saccharomyces cerevisiae and Issatchenkia orientalis) in suspension bioreactor. Firstly, it was assessed that I. orientalis is the best ITAC producer between the two species and so, further work was developed with this microorganism. Daily carbon source addition strategies led to higher ITAC titres, with the consumption of all the sugar and the reducing of the main by-product concentration, citric acid. Carbon source feeds have influence in citric acid concentration during the fermentation time. Cheese whey was used as a possible carbon-source and rich-medium supplement. The best ITAC titre was obtained in shake-flask with 50% v/v of synthetic minimal fermentation medium (MMF) and 50% (v/v) of clarified (protein-free) cheese whey. A titre in ITAC of 9.83 g/l was obtained after eight days of fermentation. With MMF in shake- flask and with fed-batch, 2.1 g/l of ITAC was obtained after five days of fermentation. Scale-up of the process from shake-flasks to liter bioreactors was done. pH control reveals to be fundamental regarding improvement of ITAC titres in bioreactor. Control of pH 4 and daily feeding of 20 g of glucose per liter of fermentation, resulted in 2.79 g/l of ITAC at day six of the fermentation. Bioreactor with 50% v/v cheese whey and 50% synthetic MMF medium led to 2.66 g/l of ITAC but a higher concentration of citric acid when compared to control (MMF only, no cheese whey).

Keywords: ITAC, suspension bioreactor, I.orientalis, protein-free cheese whey, pH control

Resumo O trabalho desenvolvido tem como objetivo encontrar as melhores condições para a produção de ácido itacónico (ITAC) usando microorganismos engenheirados (S. cerevisiae e I. orientalis) em bioreactores em suspensão. Foi testado o melhor produtor de ITAC entre as duas espécies, que se revelou ser I. orientalis, e o trabalho futuro foi desenvolvido com esta levedura não convencional. Adições diárias de fonte de carbono foram usadas como estratégia para aumentar a concentração de ITAC produzido, levando a um aumento na concentração de ITAC no meio, com o consumo de toda a fonte de carbono disponível e redução da concentração do subproduto, ácido cítrico. A concentração de ácido cítrico durante o tempo de fermentação é influenciada pela adição de fonte de carbono. Soro de leite clarificado (sem proteína) foi testado como uma possível fonte de carbono e meio rico de fermentação. A melhor concentração de ITAC foi obtida em shake-flask com 50% v/v de meio sintético (MMF) e 50% v/v de soro de leite clarificado, com 9.83 g/l obtidos após oito dias de fermentação. Com MMF em shake-flask e um sistema de fed- batch, 2.1 g/l de ITAC foram obtidos em cinco dias de fermentação. A pH 4 e com adições diárias de fonte de carbono, 2.79 g/l de ITAC foram obtidos no sexto dia de fermentação. Um bioreactor com 50% v/v soro de leite e 50% MMF levou à produção de 2.66 g/l de ITAC mas também a uma maior concentração de ácido cítrico no meio (apenas MMF, sem soro de leite).

Palavras-chave: ITAC, bioreactor em suspensão, I. orientalis, soro de leite desproteinizado, controlo de pH

Index

Acknowledgments ...... ii Abstract ...... iv Resumo ...... vi List of Tables ...... ix List of figures ...... xi Abbreviations ...... xiii 1. Introduction ...... 1 1.1 Motivation ...... 1 1.2 Objectives and challenges ...... 2 1.3 Research Questions ...... 3 1.4 Research strategy ...... 3 2. Literature Review ...... 4 2.1 Chemical synthesis and biological production of ITAC with microorganisms ...... 4 2.2 Pathways and regulation ...... 5 2.3 From modifications in natural ITAC producers to biological engineered ITAC factories..... 7 2.4 I. orientalis as a possible ITAC producer ...... 9 2.5 Fermentation Conditions ...... 10 2.5.1 Phosphate levels ...... 10 2.5.2 Nitrogen initial concentration and maintenance ...... 10 2.5.3 Oxygen supply ...... 11 2.5.4 pH and temperature ...... 11 2.5.6 Batch, Fed-Batch, and continuous fermentation operations ...... 12 2.6 Feedstocks ...... 13 2.7 Downstream strategies ...... 16 3. Materials and Methods ...... 19 3.1 Materials ...... 19 3.2 Strains used and maintenance ...... 19 3.3 Culture Medium and cultivation conditions ...... 19 3.4 Biomass determination and sugar and metabolites quantification ...... 20 3.5 ITAC production with batch fermentations of I.orientalis and S. cerevisiae in shake-flasks ...... 20 3.6 ITAC production with feed-batch fermentation of I. orientalis in shake-flasks ...... 20 3.7 Study of the toxicity of the cheese whey in I.orientalis ...... 21 3.8 Bioreactor fermentations with synthetic medium (conditions L, M, N, O) ...... 22 3.9 Bioreactor fermentations with cheese whey (conditions P, Q) ...... 23 3.10 Use of polybenzimidazole (PBI) as an adsorption polymer for ITAC and citric acid separation ...... 23 4. Results and discussion ...... 24

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4.1 ITAC production with batch fermentations of I.orientalis and S. cerevisiae in shake-flasks ...... 24 4.2 Strategies to improve ITAC and by-product decrease production in synthetic medium with I.orientalis ...... 25 4.2.1 Effect of carbon source feed regime and initial concentration on ITAC production ... 25 4.2.2 Daily sugar source and medium feed towards a fed-batch or medium renew fermentation ...... 27 4.3 Strategies to produce ITAC using cheese whey and hydrolysate as culture supplement or alternative substrate ...... 30 4.3.1 Study of the toxicity of the cheese whey in I. orientalis and S. cerevisiae ...... 30 4.3.1.3 Hydrolysis strategy to transform cheese whey in a sugar source to I.orientalis ..... 33 4.3.1.4 Daily glucose feed in synthetic and cheese whey medium ...... 35 4.4 Bioreactors ...... 37 4.4.1 ITAC production using bioreactors ...... 37 4.4.1.1 Daily sugar source feed as a precursor of a possible fed-batch or continuous fermentation ...... 40 4.4.2 Bioreactors with cheese whey ...... 42 4.5 Downstream strategy to recover ITAC production process ...... 45 5. General conclusions ...... 47 6. Future perspectives ...... 49 References ...... 51 Appendix 1 ...... 59 Appendix 2 ...... 60

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List of Tables

Table 1 - Genetic engineered microorganisms for ITAC production and titres achieved ...... 9 Table 2 - Renewable substrates available from different industries (Adapted from Banat et al. (2014) ...... 14 Table 3 - Different downstream methods applied during the years (Adapted from (Magalhães AI Jr. et al., 2016)) ...... 16 Table 4 - Summary of the ITAC and citric acid titres in the different fermentation conditions with I. orientalis. Productivity and yield were calculated for ITAC ...... 27 Table 5 - Summary of the ITAC and citric acid titres obtained in conditions A, G, H and I. Productivity and yield were calculated for ITAC ...... 31 Table 6 – Concentrations of lactose, glucose, and galactose in cheese whey before and after hydrolysis with 10M HCl ...... 33 Table 7 - Summary of all the conditions and operations tested and ITAC titre, productivity, and carbon source yield...... 44 Table 8 - pKa values for ITAC and citric acid...... 45 Table 9 – Samples of ITAC and citric acid were used in PBI for these acids recover...... 45 Table 10 - Samples of ITAC and citric acid were used in IRA458 for these acids recover...... 45

List of figures Figure 1-ITAC formation pathway in U.maydis, described by Geiser et al. (2016) – Retrieved from Geiser et al.2016 ...... 6 Figure 2- Biosynthesis pathway of ITAC (adapted from Klement & Büchs, 2013) ...... 7 Figure 3- Industrial downstream ITAC process (retrieved from Magalhães Al Jr et al (2016)) ... 17 Figure 4-Cell growth in S. cerevisiae BY4741 (blue squares) and I.orientalis SD108 (orange circles) ...... 24 Figure 5-Fermentation broth analysis for: a) I. orientalis SD108 – Glucose consumption (grey squares), ITAC (blue triangles) and citric acid (orange circles) production; b) S. cerevisiae BY4741– Galactose consumption (grey squares), ITAC (blue triangles) and acetic acid (green diamonds)...... 24 Figure 6- Shake-flask fermentations with I. orientalis using different initial sugar concentrations and feed strategies (conditions A to D). Condition A – Blue circles; B- purple squares; C – red triangles; D- Green diamonds; 1- Biomass growth in optical density; 2- Glucose consumption; 3- ITAC production; 4- Citric acid production...... 26 Figure 7- Shake-flask fermentations with I. orientalis using different initial sugar concentrations and feed strategies Condition E – blue squares; Condition F – orange circles; 1- Biomass growth in optical density; 2- Glucose consumption; 3- ITAC production; 4- Citric acid production. For glucose consumption, the glucose added daily was not measured and it is considered the theoretical value of glucose upon addition. The values for time zero and 24h after every glucose addition were measured...... 28 Figure 8-- a) MMB Ura- medium cultivated with fermentation medium from condition E; b) Shake-flasks of fermentation in condition E with I. orientalis ...... 29 Figure 9- Shake-flask fermentations with a) - I. orientalis and b) - S. cerevisiae with different concentrations of cheese whey in the medium (conditions A, G, H and I); Condition A (orange dots); Condition G (blue circles); Condition H (grey triangles) and Condition J I (yellow squares). Biomass growth in optical density...... 30 Figure 10- Shake-flask fermentations with I.orientalis with different concentrations of cheese whey in the medium (conditions A, G, H and I); ITAC concentration and glucose consumption during the fermentation time– Condition A (orange diamonds), Condition G (blue circles), Condition H (grey triangles), condition I (yellow circles)...... 31 Figure 11- Shake-flask fermentations with I.orientalis with different concentrations of cheese whey in the medium (conditions A, G, H and I); Citric acid concentration during the fermentation time in conditions A, G, H and I; Condition A (orange diamonds), Condition G (blue circles) , Condition H (grey triangles), condition I (yellow circles)...... 32 Figure 12 – ITAC (a) and citric acid (b) productions in fermentations of I. orientalis with the empty plasmid (orange) vs the fermentation with the plasmid with Atcad (blue) in a medium with 50% v/v of cheese whey and 50% v/v of MMF ...... 33 Figure 13- Analysis of the fermentation of I. orientalis in hydrolysed cheese whey at pH 4. ITAC (blue triangles), citric acid (orange circles) and glucose consumption (grey squares) ...... 34 Figure 14- Shake-flask fermentations with I.orientalis in two different conditions, E and K. Glucose consumption (grey squares) and ITAC production (Condition E- blue circles; condition K – orange triangles). For glucose consumption, the glucose added daily was not measured and it is considered the theoretical value of glucose upon addition. The values for time zero and 24h after every glucose addition were measured...... 35 Figure 15- Shake-flask fermentations with I.orientalis in two different conditions, E and K. Citric acid production (Condition E- blue circles; condition K – Orange triangles) ...... 35 Figure 16 – pH evaluation of the fermentation with I.orientalis in the conditions K and E. Photo taken at day 4 of fermentation ...... 36 Figure 17-Cell growth of I.orientalis in bioreactor with (squares) and without (circless) pH control. The control was made at pH 4 with KOH 0.5M ...... 37 Figure 18- Bioreactor fermentation with I.orientalis in conditions L and M. ITAC production (condition L – orange cicles and condition M – blue triangles) and glucose consumption (grey squares) ...... 38

Figure 19- Bioreactor fermentation with I.orientalis in conditions L and M. Citric acid production in condition L – orange circles and condition M – blue triangles ...... 38 Figure 20 – Cell viability test in conditions L (1) and M (2) for I. orientalis fermentation in bioreactor ...... 39 Figure 21- Bioreactor fermentation with I. orientalis in conditions N and O. Itaconic acid production in condition N (orange circles) and condition O (blue triangles). In both bioreactors, a 20 g/l daily feed of glucose was made. Glucose consumption and addition profile (grey squares). For glucose consumption, the glucose added daily was not measured and it is considered the theoretical value of glucose upon addition. The values for time zero and 24h after every glucose addition were measured...... 40 Figure 22- Bioreactor fermentation with I. orientalis in conditions N and O. Citric acid production in ondition N (orange circles) and condition O (blue triangles)...... 41 Figure 23 – pH levels during the fermentation time with I. orientalis in bioreactor in conditions N (orange circles) and O (blue triangles) ...... 42 Figure 24- Bioreactor fermentation with I. orientalis in condition P. Glucose consumption (grey squares), ITAC (blue triangles) and citric acid (orange circles) production during the fermentation with 50 % v/v synthetic medium and cheese whey ...... 43 Figure 25 – Bioreactor fermentation with I. orientalis in condition Q. Glucose consumption (grey squares), ITAC (blue triangles) and citric acid (Orange circles) production during the fermentation with 50 % v/v synthetic medium and cheese whey. For glucose consumption, the glucose added daily was not measured and it is considered the theoretical value of glucose upon addition. The values for time zero and 24h after every glucose addition were measured. 43

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Abbreviations

AtCAD - Cis-aconitate Decarboxylase gene from Aspergillus terreus

Abs – Absorbance

ALR- Air-Lift Bioreactor

CAD – cis-aconitate decarboxylase enzyme

CSM - Complete Supplement Mixture

DO – Dissolved oxygen

FBA – Flux Balance Analysis

FDA - Food and Drug Administration

GMO- Genetically modified organism

GRAS – Generally Recognized as Safe

HPLC – High-performance liquid chromatography

ITAC – Itaconic acid

Km – Michaelis constant

JSC – Jatropha seed cake

MMA – methyl methacrylate

MMB – minimal growth medium

MMF – minimal fermentation medium

NP – Natural producer

NRRL- Northern Regional Research Laboratory

OD – Optical density

PFK – phosphofructokinase

RFD - reverse-flow diafiltration

SBR - styrene-butadiene rubber

STR – Stirred-Tank Bioreactor

TCA – tricarboxylic acid cycle

USA – United States of America

USDA - United States Department of Agriculture

YNB – Yeast Nitrogen Base

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1. Introduction

1.1 Motivation Itaconic acid (ITAC) was recognized in 2004 by the United States Department of Energy as one of the 12 top molecules with potential to be produced through a biological route (Werpy et al. 2004). However, ITAC can also be produced by chemical synthesis. To be economically competitive the bioprocess should achieve productivity levels of 2.5 g. l-1.h-1 obtained with chemical synthesis, value that needs to be accoupled with the decreasing of the production costs, that include all the upstream, process and downstream areas.

ITAC is a dicarboxylic organic acid that was first mentioned by Baup in 1837 (Karaffa et al. 2015; Kuenz et al. 2012; Willke & Vorlop 2001) as a product of citric acid distillation. Its acidic capabilities are related with its pKa values. The pKa values fluctuate in the literature, with the most part of the information pointing for pKa values of 3.84 and 5.55 that mark the transition between the three different protonation states. This methylene succinic acid with molecular formula C5H6O4 has molecular weight of 130.10 g/mol, boiling point of 268 ºC and freezing point of 165-168 ºC (tests are variable for this parameter). Regarding the solubility, it is very soluble in water and soluble in benzene, chloroform, ether, carbon disulfide, alcohols, and petroleum ether (parchem, Sigma- aldrich, pubchem). In its solid state, ITAC is a white crystalline powder and is a naturally occurring compound, non-toxic and readily biodegradable (Miami chemical).

A study (Weastra report, 2011) for the market potential of some selected chemicals establishes an ITAC market with a value of $74.5 million and 41 400 tons in 2011. As a main application, identified at that time, 44% of the global ITAC market was estimated to be in the production of styrene-butadiene rubber (SBR) latexes. With a price of $1800 to $2000/ton, it is a need to reduce the production costs so that the final cost can decrease and increase the demand for ITAC. Regarding the potential of ITAC in targeted applications and in other possible bio-based routes and intermediates, the projected market for ITAC is estimated to approximately 407 790 tons with the value of approximately $567.4 million in 2020 (Weastra report, 2011).

The production of ITAC is mainly concentrated in China nowadays, where the production costs are lower. Another important aspect is that the rules regarding bioprocess toxicity using Aspergillus terreus due to pathogenic toxicity (Fernández et al. 2013; Lass-Florl et al. 2005; Steinbach et al. 2004), are almost none which contrast with the tight rules imposed by the other countries about toxicity in manufacturing. No information about A. terreus being considered as GRAS (Generally Recognized as Safe) was found in FDA (Food and Drug Administration). However, USA and France are examples of countries with facilities to re-produce ITAC, i.e., to start to produce ITAC again since they were already producers.

ITAC is widely used in the industry, especially in the polymers industry. It can participate in addition polymerization, since of the advantage of the double bond a two carboxyl groups of ITAC. Its versatility makes it available to be used as a co-monomer to form heteropolymers. El-Imam

AA & Du C review (2014) summarized quite well the current applications of ITAC and the polymers made with this building block in today’s market. Some products of ITAC polymerization reactions are: poly-ITAC (PITAC or PIA) that is a polymer of only ITAC monomers; SBR latex that is a mixed polymerization of styrene, butadiene and ITAC (here it works as a co-monomer); carpets, water treatment, adhesives, deodorants, reinforced glass fiber, ion-exchange resins, fabric blinders, paints, and artificial jewelry. Robert & Friebel review (2016) summarized the polyesters derived from ITAC, emphasizing that one of the best characteristics of this carboxylic acid is its interesting chemical structure. ITAC has two carboxylic groups and an additional double bond that allows more reactions to be done, as functionalization reactions or cross-linking. This makes ITAC a possibility to be widely use in crosslinking applications, such as drug delivery, shape memory polymers, elastomers, and coatings, achieving results with equal or higher quality in comparison with standard crosslinking materials.

ITAC can be also used to produce methyl methacrylate (MMA), being this a huge potential market for this carboxylic acid. MMA is currently produced using acetone cyanohydrin. However, ITAC can be a substitute for this reagent in MMA production (Weastra Report, 2011). MMA is nowadays the top ester of methacrylic acid that has interesting capabilities as its good strength or transparency, as well as an excellent weather resistant. With an outstanding set of applications, MMA is an interesting market for the development of green routes for its production in opposite to the chemical way, as Lucite International is already exploring with patents on the field, with a projected market volume of 1 664 tons and $3 billion based on 2011 market sizes. (Weastra Report, 2011).

If these suggested substitution markets are reached, the demand for ITAC will augment widely and the production needs to be much higher than currently. This opens a door for new producers (or re-producers), new investments, and new discoveries to achieve the more efficient and more profitable conditions possible with the cleanest ITAC producer.

1.2 Objectives and challenges In the present study there are some objectives established from the beginning and objectives that were proposed during the process according to the results obtained. As described in the literature, the industrial and commercial production is made with fungus in submerged fermentation, which makes cumbersome the bioreactor operation, control of culture conditions and uses a non-GRAS microorganism. The use of filamentous fungus is a problem in bioreactors, and new organisms need to be tested to solve this problem. The use of an engineered and GRAS microorganism for ITAC production was one of the main objectives of this work. Starting with this microorganism, the search for strategies that could improve the titres and carbon yield of ITAC using suspension bioreactor production system is a mandatory aim, as it simplifies scale-up, feed of substrate and downstream. The production of ITAC by I. orientalis, one of these engineered and GRAS microorganisms possibility (Holban & Grumezescu 2017), with bioreactors is not reported in the literature, which gives to this work another important objective, to understand the behavior of this non-conventional yeast in suspension bioreactor for ITAC production. Following this, one of the

2 major challenges of this work is to find a strategy capable of establish I. orientalis as a potential ITAC producer for industry, studying the effect of pH control and substrate. The use of agro- industrial residues as carbon source or complex medium supplements is important in the economy of the process. In this study, cheese whey was used as an example of an agro-industrial residue, aiming to replace crystalline glucose that is expensive and then decrease the production cost and so, the selling price. Transformation of cheese whey from a waste to be used as carbon source or rich medium is other of the objectives of this study. The isolation of ITAC from the fermentation broth and its by-products (or the elimination of them during the fermentation) is the last of the initial proposed aims for this project.

1.3 Research Questions • What is the best ITAC producer between genetically modified S. cerevisiae BY4741 and I. orientalis SD108, concerning ITAC titre and productivity? • What is the effect of adding cheese whey in S. cerevisiae and I. orientalis cultures concerning ITAC production? • What is the effect of pH control and carbon source feeding in ITAC production when cultured in 1L bioreactor? • Can the genetically modified S. cerevisiae and I. orientalis cultures for ITAC production be scaled-up from 50 mL shake-flask to 1L bioreactors? • Which by-products will be produced during the process? Can operation conditions be selected to reduce by-product concentrations? • Can ITAC be isolated from the fermentation broth using an adsorption process?

1.4 Research strategy To achieve the objectives and answer the research questions, this study made use of:

• Microorganisms: S. cerevisiae BY4741 and I. orientalis SD108 were used as cell factories for ITAC production. Comparison was made which of the two species was a higher ITAC producer to further studies. • Different strategies were tested to achieve the best ITAC titre, including the use of different carbon source feed strategies, different initial carbon source concentrations and the substitution of medium and not only carbon source. • Cheese whey was tested to be used as a carbon source or rich medium for ITAC production by these microorganisms. Evaluation of cheese whey was done regarding the sugars and metabolites present. • Bioreactor tests were made based on batch and fed-batch fermentations, with and without control of pH with synthetic and cheese whey as an alternative medium. • Polybenzimidazole (PBI) was used as an adsorption platform for ITAC and fermentation by-products separation.

2. Literature Review 2.1 Chemical synthesis and biological production of ITAC with microorganisms As reported in the last section, ITAC was firstly named by Baup as a product of citric acid distillation. Until the 60’s in XX century, chemical synthesis was the most common method for ITAC production. These chemical methods used for ITAC production include: dry distillation of citric acid and treatment of the anhydride with water (Willke & Vorlop 2001), heating of calcium aconitate solution produced in the cane sugar refining process (El-Imam AA & Du C, 2014), Montecatini method involving propargyl chloride (El-Imam AA & Du C, 2014; Willke & Vorlop, 2001), oxidation of mesityl oxide and isomerisation of the citric acid (Berg & Hetzel, 1978) and the oxidation of isoprene (Pichler et al., 1967).

Meanwhile, in 1932, Kinoshita reported the bioproduction of ITAC by fungi from carbohydrates in Aspergillus Itaconicus (Kinoshita 1932), being the first example of production of ITAC by biological route, and so, with microorganisms. After this discovery, interested was generated for the discovery of other microorganisms able to naturally produce ITAC and with the possibility of achieving better titres than A. itaconicus. In 1939, Calam et al. found a new strain capable of being a natural producer of ITAC, whose name is Aspergillus terreus (Calam et al. 1939). The Northern Regional Research Laboratory (NRRL) of the United States Department of Agriculture (USDA) screened different wild type strains for ITAC production and identified A. terreus NRRL 1960, as the most competent ITAC producing strain (Lockwood & Reeves 1945). A. terreus is nowadays the mostly used industrial producer of ITAC. Its usage started in 1945, when Pfizer & Co., Inc. established submerged fermentation of A. terreus for ITAC production as an industrial production system (Kane et al. 1945). However, this strain is very sensitive to fermentation conditions, as agitation and aeration or substrate impurities, that have influence in the growth of A. terreus. A. terreus is known to produce extremely toxic compounds that can affect human health and this fungus is related with diseases as invasive aspergillosis (Hachem et al. 2004). Another important task was to select a ITAC production microorganism towards final composition of the fermentation broth with a lower ratio of by-products to ITAC.

In the exploration for these new microorganisms, Haskins et al. (1955) found ITAC in the fermentation broth of an Ustilago zeae (then recalled Ustilago maydis) strain. Some years later, the Iwata Corp., a Japanese company with interest in ITAC production, screened different Ustilago species including the U. maydis and found a production of 53 g/l of ITAC in 5 days (productivity of 0.44 g. l-1. h-1) using glucose as substrate (Tabuchi, 1991).

However, cultivation of filamentous fungi are a problem in bioreactor fermentations (García-Soto Mariano 2006). Fungi can grow as free mycelia or in pellet form. In the first situation, the viscosity increases, inducing a significant reduction on mixing and air dispersion and thus lead to a decrease in the oxygen availability for cells in growth medium. In the second case regions with

4 different growth patterns and substrate availability, which will affect the fermentation yield and biomass growth. To address this problem, yeasts were also tested for ITAC production.

Tabuchi, in 1981, screened more than 140 strains of yeasts to produce organic acids from glucose. The strain S-10 from Candida was found to produce ITAC from glucose with almost 35 g of ITAC produced per 100 g of glucose during five days of fermentation (productivity of 0.29 g. l-1. h-1). Rhodotorula species were also tested for ITAC production but reached only 15 g/l of ITAC from glucose after 7 days, which corresponds to a productivity of 0.089 g. l-1. h-1) (Kawamura et al.,1981).

2.2 Pathways and regulation The discussion on the elucidation of the metabolic pathway for ITAC production was not always consensual. Kinoshita first suggested that the decarboxylation of aconitate is the key reaction and so, involving glycolysis and tricarboxylic acid cycle (TCA) in ITAC production (Kinoshita, 1932). This suggested pathway for ITAC production basically states that a sugar, per example glucose, enters in glycolysis pathway to be converted into pyruvate. Then, in the first steps of the citric acid cycle, citrate and cis-aconitate are formed. As a last step, the only step dedicated exclusive to ITAC production, the cis-aconitate decarboxylase enzyme (CAD) converts cis-aconitate into ITAC by releasing a carbon dioxide molecule.

However, some studies questioned the involvement of the TCA cycle and suggested an alternative pathway for ITAC production from pyruvate (confirmed as an intermediate) to ITAC. Walker in 1949 (Shimi, 1962) suggested ITAC formation from the condensation of two pyruvate molecules with prior dehydration and after an oxidative decarboxylation. Bentley and Thiessen confirmed with labelled substrates the pathway suggested by Kinoshita. Eimhjellen and Larsen, in 1955, were not able to observe the conversion of citric to ITAC and so, they were not able to support the work developed by Bentley and Thiessen. Shimi, in 1962, suggested a reaction of acetyl CoA and phosphoenol pyruvic acid to be the base of ITAC production. In the 90s of the XX century, (Jaklitsch et al. 1991) and (Bonnarme et al. 1995) investigated the suggested pathways for ITAC production from pyruvate that had been discussed over the years. After this investigation, the authors suggest that the pathway proposed by Kinoshita is the one that is likely to be correct.

Another important discovery was that CAD is not located in the mitochondria but in cytosol (Jaklitsch et al. 1991). In this test, ten enzymes related with the ITAC biosynthesis (showed in Figure 2) were studied, showing that CAD is the only one present in the cytosol in opposite with the enzymes preceding in the pathway in TCA cycle, that were found in mitochondria. In the same study it is proposed by the authors that cis-aconitate is transported by malate–citrate antiporter into the cytosol where is then converted to ITAC. However, no experimental results confirm this suggestion which opened the door to new perspectives about mitochondrial carriers for cis- aconitate (Jaklitsch et al. 1991).

Bonnarme tested in 1995 the ITAC biosynthesis pathway, making use of 14C and 13C-labelled substrates and nuclear magnetic resonance spectroscopy. The authors state that there is no doubt that TCA cycle is part of ITAC biosynthesis.

The enzyme CAD was isolated and characterized by (Dwiarti et al. 2002), and it became an important step for the understanding of ITAC biosynthesis. Some important properties of the A. terreus Cad (AtCad) enzyme were determined including the KM value of 2.45 mM (37º C, pH 6.2) and that its activity drops significantly at pH 7.5, in values more than 80% inferior to the maximum value obtained. In 2008, the responsible CAD gene in the genome of A. terreus (AtCAD) was finally identified (Kanamasa et al. 2008).This discover open the door and allowed the transformation of non-natural ITAC producer into producers. S. cerevisiae was transformed with this AtCAD gene, confirming CAD as the enzyme responsible for the ITAC production. A transcriptional approach of (Li et al. 2011a) recently supported these findings.

The importance of the enzyme activity and expression was also studied by Kanamasa in the same study, where five times higher transcription levels of AtCAD led to higher ITAC production. Since no change in the amino acid sequence was detected, the activity and transcription levels of the Cad are then crucial for the performance of the ITAC biosynthesis pathway. Blumhoff et al. (2013) showed the same situation in Aspergillus niger under various constitutive promoters of different expression strength. These discoveries opened the door to the next step in this process, the modification of microorganisms to produce ITAC.

Recently, Geiser et al. (2016) found that U. maydis uses trans-aconitate and not cis-aconitate to produce ITAC. This means that instead of CAD, U. maydis uses trans‐aconitate decarboxylase (TAD1).

Figure 1-ITAC formation pathway in U.maydis, described by Geiser et al. (2016) – Retrieved from Geiser et al.2016

Figure 2- Biosynthesis pathway of ITAC (adapted from Klement & Büchs, 2013)

2.3 From modifications in natural ITAC producers to biological engineered ITAC factories As different organisms were tried, some authors decided to choose a different strategy to improve ITAC production yields. Instead of using only the wild-type strains for ITAC production, the authors decided to use different and innovative strategies. One of these strategies used in ITAC producing strains was mutagenesis. Starting from the wild-type strains, the authors looked for mutations that could be benefic for ITAC production, per example modifying genes related with the TCA cycle. A Candida mutant produced up to 42 g/l of ITAC after 6 days, with a productivity of 0.29 g. l-1. h-1 (Hashimoto et al.,1989), increasing in 7 g/l the result obtained in 1981 by Tabushi, while mutation of A. terreus IFO-6365 in Japan led this mutated strain (TN-484) to produce 82 g/l of ITAC after 7 days with 160 g/l of glucose, and so with a productivity of 0.48 g.l-1.h-1 and a carbon yield of 0.51 (Yahiro et al. 1995). Levinson studied fourteen yeast strains never characterized for organic acid production, including Pseudozyma antartica. One of the strains tested, P. antartica NRRL Y- 7808 was found to produce 30 g/l of ITAC from 80 g/l of glucose in shake-flask cultures under limiting nitrogen conditions, with a yield of 0.375 and a productivity of 0.11 g. l-1.h-1.

After the discovery of Kanamasa in 2008 that targeted the gene responsible for CAD enzyme synthesis in A. terreus, another strategy was proposed in the literature to genetically modify species and strains not able to produce ITAC naturally and make them non-natural producers. Li et al. successfully developed A. niger transformants and optimized medium conditions to produce ITAC in this specie (Li et al. 2011). Using a clone-based transcriptomics approach, a key gene of the ITAC pathway, AtCAD, was identified and the expression of this gene in Escherichia coli as an expression vector, resulted in ITAC production, confirming its identity as cis-aconitate decarboxylase (first time confirmation by a clone-based transcriptomics approach). After this confirmation, A. niger AB 1.13 was transformed with a plasmid with the AtCAD gene for ITAC production, with the best transformant achieving 0.7 g/l of ITAC from 62.9 g/l of glucose, with a carbon yield of 0.011. A. niger was selected as host microorganism because it has a regular use in industrial biotechnology, for example in the production of pectinases and citric acid.

Recombinant production microbiological hosts such as Escherichia coli used in Li work (2011) have been proposed for cheaper production of ITAC (Yu et al. 2011). E. coli, a facultative anaerobium organism it has many advantages as production host, namely rapid growth and well- established protocols for genetic modifications (Vuoristo et al. 2015). Harder et al. used a modified strain of E. coli with the plasmid pCadCS in a fed-batch strategy and achieved 32 g/l of ITAC, with a carbon yield of 0.49 and a productivity of 0.45 g.l-1.h-1 (Harder et al. 2016).

S. cerevisiae was also engineered for ITAC production. Blazeck et al. tested S. cerevisiae and then subsequently deleted ade3, bna2, and tes1 genes to look for an improvement in ITAC titre and achieved 168 mg/l, with a productivity of 0.002 g.L-1.h-1 (Blazeck et al. 2014). S. cerevisiae has its genome completely sequenced which enable metabolic engineering approaches. At iBB- IST, Campos (Campos, AVS, 2016) used metabolic modulation based in Flux Balance Analyses (FBA) to identify 21 genetic deletions with the potential to ITAC production in S. cerevisiae. The increased production was then confirmed for 6 of the 21 deletions proposed.

Yarrowia lipolytica, a fungal species in the Dipodascaceae family was also engineered for ITAC production by Wang and Blazeck (Blazeck et al. 2015; Wang et al. 2011). Y. lipolytica was evaluated by the authors as an excellent potential candidate for ITAC production due to its capacity to accumulate citric acid cycle intermediates and its tolerance to lower pH. Wang et al. obtained an ITAC concentration of 2.6 g/l from 100 g/l of glycerol at pH 4, which means a yield of 0.026. Blazeck et al. produced ITAC in Y. lipolytica through heterologous expression of gene that codifies for the ITAC synthesis enzyme, AtCAD, which resulted in an initial production of 33 mg/l from 20 g/l of glucose, with a yield of 0.0016. However, the authors optimized the strain via metabolic pathway engineering, enzyme localization, and medium which enabled a production in bioreactors of 4.6 g/l but with 80 g/l of glucose with pH 3.5, with a yield of 0.057.

The highest achieved concentration in the literature was published by Hevekerl et al. (2014) achieving 146 g/l of ITAC after fermentation optimization with A. terreus (natural producer) and with pH in this work and previously found optimizations, so the results for genetically modified microorganisms are not so significant when compared to natural producers. However, in 2017,

8 the same group was able to produce 160 g/l from A. terreus, controlling the pH at 3.4 over the fermentation time (Krull et al. 2017), becoming this the highest achieved titre of ITAC. The production titre of some genetically engineered and natural producers is showed in Table 1. For these engineered microorganisms, the higher value achieved is 4.6 g/l with Y. lipolytica, with the results for the other testes microorganisms, except for E. coli, around 100 mg/l.

Table 1- Genetic engineered microorganisms for ITAC production and titres achieved

Microorganism Author ITAC concentration

A. terreus Kane et al.,1945 27 g/l A. terreus Hevekerl et al. 2014 146 g/l A. terreus Krull et al. 2017 160 g/l P. antartica Levinson et al. 2006 30 g/l E. coli pET-9971 Li et al., 2011 < 1 g/l Liao and Chang, E. coli BW25133 4.1 g/l 2010 A. niger AB 1.13 Calam et al., 1939 0.6 g/l Y. lipolytica Wang et al., 2011 2.6 g/l Y. lipolytica Blazeck et al., 2015 4.6 g/l E. coli Harder et al. 2016 32 g/l S. cerevisiae 168 mg/l (with high initial BY4741 (Δade3, Blazeck et al., 2014 DO) Δbna2, Δtes1)

2.4 I. orientalis as a possible ITAC producer Issatchankia orientalis as lastly renamed by Kurtzman in 2011 (or Candida Krusei, Pichia kudriavzevii) is a non-conventional yeast reported to be an ethanol thermo and organic acid tolerant producer (Xiao et al. 2014). It has been isolated from natural sources such as fruits, namely from the orange and grape juice. In 2014, Xiao and colleagues reported the use of this yeast as a succinic acid producer, suggesting the potential modification of this strain to become a ITAC producer by the introduction of the plasmid with the AtCAD gene (Xiao et al. 2014). In this work, different carbon sources were tested, with the conclusion that fructose and glucose are the most suitable to be consumed by I. orientalis. After the engineering of the strain, about 11.63 g/l of succinic acid was achieved, at carbon yield of 0.12. No other study was found in literature using I. orientalis for ITAC or other organic acid production, which makes it interesting to be addressed. Within this thesis this strain is used as host to produce ITAC after AtCAD introduction.

2.5 Fermentation Conditions The fermentations conditions selected in different studies, both in shake-flasks or bioreactors, can limit the higher production of ITAC titres as the fact of using a microorganism with an inserted plasmid needs different conditions for an efficient process, since as an example, some plasmids show sensitivity concerning temperature. Nutrients may drive the metabolism towards metabolic pathway that favors ITAC production. This section briefly summarizes the conditions, previously assessed for ITAC production, highlighting the most relevant and important aspects.

2.5.1 Phosphate levels According to some authors, phosphate limitation is required for A. terreus to produce ITAC (Welter, 2000; Willke & Vorlop, 2001). In work from Welter and for A. terreus NRRL 1963, ITAC production started after phosphate depletion to levels below 1 mg. l-1. The explanation for this phenomenon can be related with the fact that low phosphate concentrations have interference in the decrease of ATP levels and the global energy of the cell, thus leading to an accelerated flow through glycolysis (Klement & Büchs 2013).

Kuenz et al. 2012 varied the concentration of the phosphate source (KH2PO4) from 0.04 to 0.16 g/l using A. terreus DSM 23081, that are concentrations 40 and 160 times higher than that used by Welter, 2000. Kuenz used low phosphate concentrations, but with the highest one used in comparison with Welter, to show that ITAC production increases with the higher phosphate concentration. This study suggests that low phosphate concentration, but not completely depletion of it is required for ITAC production. When the process was scaled up for 15 L glass stirred tank bioreactor with 180 g/l of initial glucose and initial pH of 3.1, 86.2 g/l of ITAC were produced. Phosphate concentration decrease was observed during the growth phase was observed from about 62 to 3.36 mg/l over 2.6 days. This is representative that the phosphate is used for the growth of the organism, and then, the ITAC production starts when these phosphate levels are very low.

Hevekerl et al. (2014a) showed that the limitation of phosphate did not led to a higher production of ITAC, and in opposite, an increasing of this concentration from 0.1 to 0.8 g/l led to the higher ITAC produced by the authors, with a higher biomass growth.

2.5.2 Nitrogen initial concentration and maintenance Information about nitrogen levels on ITAC production fermentation medium are disperse. Papagianni et al. (2005) stated that ammonium may prevent the citrate inhibition of the phosphofructokinase (PFK), an enzyme from the glycolytic pathway, enhancing the glycolysis flux to the TCA cycle, even in the presence of citrate. Vassilev (1992) discovered that the best production rates were achieved in immobilized A. terreus cells when no nitrogen sources were added, in comparison with the addition of ammonium nitrate. In other microrganisms, like U. maydis or P. antartica, nitrogen limited conditions led to the production of ITAC even at moderate pH levels (Klement et al. 2012; Levinson et al. 2006). Kuenz et al. (2012) measured ammonium and nitrate concentrations in 1.5 L stirred bioreactor. Their studies showed that an

10 initial reduction of 50% of nitrogen concentration produced similar results to the control. However, when this concentration was lowered to 10% of the control nitrogen concentration, the behavior was different and not beneficial for ITAC production. Pais, D. (Lab Report) tested the impact of doubling the nitrogen sources available for ITAC production in S. cerevisiae BY4741. Higher nutrient availability led to higher growth and ITAC produced.

2.5.3 Oxygen supply In the biochemical point of the view, oxygen availability is an important factor for the TCA cycle to work. Oxygen is not needed in the TCA cycle itself, but it is fundamental for the regeneration of NAD+ from NADH. NAD+ is a key factor in many reaction of TCA cycle. So, a balanced oxygen supply is fundamental for ITAC production since anaerobic conditions can irreversibly damage the biomass and shut-down TCA cycle. To produce ITAC, 1.5 mol of O2 are consumed per mol of ITAC produced from sugar source (Klement & Büchs, 2013). One of the examples of the oxygen importance in ITAC production is the test with A. terreus where a severe lack of oxygen supply caused by turning off the aeration, damage irreversibly the ability of A. terreus cells to produce ITAC (Welter, 2000) and so, the bioreactor must be well aerated and mixed. On the opposite side, with A. terreus, the increasing of stirring to input more oxygen in the medium has the problem of the mechanic stress that the higher stirring applies to the cells. Experiment with A. terreus DSM 23081 indicated that an interruption of the oxygen supply for 10 minutes has an influence on the morphology and causes a dramatic decrease of ITAC productivity as well as ITAC end concentration (Kuenz et al., 2012). These results seem to be comparable to other literature results with A. terreus NRRL 1960, where interruptions of the oxygen supply for 5 or 10 minutes affected the ITAC production dramatically and the production only re-starts slowly after 24 h (Gyamerah 1995). (Li et al. 2011; Li et al. 2013) tested different levels of dissolved oxygen for both A. terreus and A. niger, observing that a lower oxygen supply (25 % of DO) was optimal for ITAC production in controlled batch fermentation using A. terreus and also that lower DO conditions (10–25 %) eliminated oxalic acid accumulation as a by-product.

While for filamentous organisms such as A. terreus, there is always a conflict between sufficient oxygen transfer and cell damage by hydromechanical stress as explained above, new organisms tested for ITAC production as E. coli, S. cerevisiae or another non-conventional yeast, tolerant to higher shear stress are welcome for the scale-up of the process.

2.5.4 pH and temperature The influence of the pH on the production of ITAC was investigated several times. In the cultivation of A. terreus most of the studies focused either on variations of the initial value of pH or pH control during cultivation. Rychtera & Wase (1981) discussed the importance of pH and stated that in both batch and continuous fermentation it is one of the significant parameters. However, the highest ITAC titre reported in the literature until 2014 was 86.2 g/l (Kuenz et al. 2012), and this result was achieved without pH control. Values of pH 2 are typically reported in the literature (Park et al. 1994; Yahiro et al. 1995) but higher values as pH 5.9 by Gyamerah (1995) are also reported. (Riscaldati et al. 2000) achieved the higher ITAC concentration when

11 the pH was kept at pH 2.8, while Rychtera & Wase (1981) obtained higher product formation at pH 2.1 to 2.2. However, in 2014 (Hevekerl et al. 2014) did not observe that the initial pH showed differences on the product formation for initial pH between 3.1 and 4.9. ITAC production was lower when pH control took place from the beginning of the fermentation, compared with the non- controlled pH. The increase in the final ITAC concentration occurred only when a pH control was initiated after 2 days of cultivation. The authors could raise the final ITAC titre to 146 g/l, an increase of 62% to the result obtained by Kuenz et al. (2012). The pH shift led to a reduction of by-product formation with the concentration of the by-products reduced from 3.3 % to a maximum of 2.1 % in the total of organic acids in fermentation broth. The decrease in the by-product formation after a pH shift was also reported by (Mario & Schweiger, 1963). (Eimhjellen & Larsen 1955) observed that non-proliferated mycelia need an acidic pH to be able to produce ITAC, as when they were in medium with pH 2, ITAC was produced but in pH 6 no ITAC was found in the fermentation broth. Blazeck et al. (2015) also achieved better results for ITAC production with Y. lipolytica when the pH was controlled at 3.5. More recently for A. terreus, when pH was controlled at 3.4, 160 g/l were obtained (Krull et al., 2017).

Temperature was also studied during the years for the best performance of the fermentation. (Yahiro et al. 1997) report that the optimum temperature for both growth and ITAC formation is 37 °C for A. terreus. In 2012, Rafi et al. (2014) tested different temperatures ranging from 25 to 37 °C in U. maydis with the best result when temperature was at 32 ºC. In 2014, (Hevekerl et al. 2014) tested different fermentation temperature in a range of 31 to 37 °C. The highest ITAC concentration of 76 g/l was achieved at 35 °C while the product concentrations at 33 and 37 °C were quite similar, being 66 and 65 g/l of ITAC, respectively. The lowest concentration of 55 g/l of ITAC was found at 31 °C.

2.5.6 Batch, Fed-Batch, and continuous fermentation operations Batch fermentations for ITAC production have been made along the years in shake-flasks, Stirred Tank Reactor (STR) and Air-lift Reactor (ALR). As the name say, in the STR, the fermentation medium and cells are placed into the tank and the medium is stirred during the fermentation. In the opposite, in ALR the medium is circulated and mixed using pressurized air and it is a good alternative when shear sensitive strains are being used. One of the advantages of the ALR is that it uses one third of the energy compared with STR. Using different A. terreus strains along the years, the best overall result in ITAC concentration and productivity, with 146 g/l and a productivity of 1.15 g.l-1 .h-1 was achieved in a 1.5L parallel bioreactor system (DASGIP and conditions monitored using DASGIP Control software) with A. terreus DSM 23081 (Hevekerl et al. 2014). In Kuenz et al. (2012) work, a total 86.2 g/l of ITAC and 0.51 g.l-1.h-1 productivity in STR (Biostat E, Braun Biotech International GmbH, Germany) was obtained with the same strain. ARL was tested by (Okabe et al. 1993) and Yahiro et al. (1997), in this case using A. terreus IFO 6365 and TN- 484, respectively. The best result was achieved by Yahiro et al. (1997) with 63.7 g/l of ITAC and obtained with a productivity of 0.64 g. l-1.h-1. Both made use of the same bioreactor system.

Repeated Fed-Batch was used in many articles published. In this type of fermentation, the volume changes with the fermentation time due to the substrate addition. Both free-cells and immobilized cells were used. (Park et al. 1994) used both shake-flasks and ALR for ITAC production. As in other cases, the shake-flask showed an increased ITAC concentration, productivity and yield when compared with the ALR. This might be due to oxygen limitations in the ALR since mixing is milder than that in the rotary shaker.

Kautola et al. (1990, 1991) tried different immobilization hypotheses in flasks, as agar gels, calcium alginate, celite R-626 or polyurethane foam cube. The first three strategies showed poor results while the last one showed 51 g/l of ITAC produced with a productivity of 0.15 g.l-1.h-1 (Kautola et al., (1985; 1990; 1991).

Recently and as already addressed in this review, Harder et al. used a modified strain of E. coli with the plasmid pCadCS in 1L Multifors-bioreactors (Infors) and a glucose starting concentration of 27 g/l and a fed-batch of glucose everytime the glucose levels went down to 10 g/l. With this strategy the authors achieved 32 g/l of ITAC and less than 5 g/l of acetate and pyruvate as by- products.

Continuous fermentation to produce ITAC is not a common strategy used. Kobayashi & Nakamura (1966) and Rychtera & Wase (1981) work in a continuous fermentation with free-suspended cells. The productivity for both processes is under 0.5 g. l-1.h-1. Ju & Wang (1986) used disk bioreactor with A. terreus NRRL 1960 grown and immobilized on porous disks. Basically, the mycelia of A. terreus were grown in these disks but the process is too slow, and it requires the rotating in and out of the medium. From glucose, the authors could achieve 18.2 g/l of ITAC with a productivity of 0.73 g. l-1.h-1. Kautola et al. (1990) achieved 15.6 g/l with column bioreactor and immobilizing A. terreus in polyurethane foam cube.

Recently, Carstensen (Carstensen et al., 2013) used a continuous fermentation process using U. maydis. however, their aim was to find a better downstream strategy and not focus on optimized media or fermentation conditions

2.6 Feedstocks During the last decades, many efforts were carried out aiming to reduce the overall costs of fermentation for ITAC production. One of the proposed strategies is reducing costs with substrates, using low value feedstocks. In most of the research and development assays, it is used commercial crystalline glucose as carbon source, which is very expensive for this kind of process. Still, it is with glucose that the researchers achieved better ITAC titers. Nowadays, there are also several researchers focused on using cheap feedstocks for fermentation tests, such as industrial wastes or plant biomass. Agricultural residues such as cereal straws, corn starch and wheat straw, forest residues such as hardwoods, and industrial wastes or by-products such as glycerol and cheese whey have thus become potential sources for cost-efficient microbial production of organic acids (Alonso et al. 2015). In this chapter, the used substrates used for ITAC production are addressed.

Banat et al. ( 2014) presented a list of renewable substrates available from different industries (Table 2)

Table 2-Renewable substrates available from different industries (Adapted from Banat et al. (2014)

Waste/residues with potential to be Industry used as fermentation substrates Agro-industrial waste Beet Molasses, rice, straw of rice Animal fat Waste Coffee processing residues Coffee pulp, coffee husks Crops Potato, sweet potato, soybean Dairy Industries Curd whey, cheese whey, whey waste Food processing industry Frying edible oils and fats, olive oil, sunflower Fruit processing industry Banana waste, Pomace of apple and grape Oil processing mills Cake, soapstock, waste from lubricating oil

The use of beet or sugarcane molasses was firstly proposed by Kane et al. (1945). In 1964, Nubel & Ratajak used beet and sugarcane molasses pre-treated by ion-exchange or ferrocyanide for ITAC production. Sugarcane molasses are a by-product from the sugar purification process and have a high carbohydrate content and with this characteristic, it is seen an attractive source to produce organic acids.

Corn starch is another useful and cost-effective carbon source, easily accessible at relatively high purities (Reddy & Singh, 2002). Corn starch has a high potential as effective carbon source for ITAC production, with over 60 g/l of ITAC obtained by A. terreus TN-484 in a 2.5 L ALR bioreactor using 140 g/l of corn starch hydrolyzed with nitric acid without any additional nitrogen source (Yahiro et. al, 1997). However, its ability to gelatinize upon heating makes it difficult to sterilize and so, a pretreatment with acid to hydrolyze corn starch needs to be done.

Okabe et al., (2009) also hydrolyzed the starch, which gave ITAC yields of 0.36 g ITAC/g starch while Dwiarti et al. (2007) was able to perform fermentation with A. terreus from starch materials such as corn and sago starch hydrolysates, with a productivity of 0.4 g. l-1.h-1 in both cases. Interestingly, the strategy used for the hydrolyzation has influence on the requirement of nitrogen source, as described by Okabe, where for corn starch hydrolyzed with enzymes there is no requiring for additional nitrogen source, but for sago starch, additional nitrogen source is required.

Kobayashi proposed in 1978 a process to produce ITAC from hydrolysate of wood waste (Kobayashi, 1978). Studies made by Kautola on A. terreus (Kautola et al., 1985) and Klement on U. maydis (Klement et al., 2012) revealed that this microorganism can use xylose from the hemicellulosic fraction present in wood. However, Kautola proposed that the both cellulose and hemicellulose fraction of the wood should be used as substrate for the process since when xylose was used, the fermentation process exhibited lower ITAC yields. Tippkötter et al, (2014) used

14 wood hydrolysates for ITAC production in A. terreus as part of the German lignocellulose biorefinery project (Michels, J., & Wagemann, K., 2010). However, the wood hydrolysates seem to have some compounds that inhibit the growth and ITAC production in A. terreus. Another objective of the German lignocellulose biorefinery project was the usage of grass silage and its juices to produce ITAC (Klement & Büchs 2013).

Glycerol is other of the possible feedstocks for ITAC production, with glycerol itself, and mixtures of sucrose and glycerol tested as substrates, resulting in high ITAC yields obtained and glycerol fully consumed (Jarry & Seraudie 1997). However, the presence of impurities such as methanol, salts, free fatty acids, and free methyl esters in crude glycerol limits the microbial growth and fermentation performance (Alonso et al. 2014).

In 2006, Rao et al. reported that Jatropha curcas seed cake (JSC) is one of the best carbon sources among various carbohydrates for ITAC production. J. curcas is a plant of the Euphorbiaceae family, was firstly found in central America and used to produce biodiesel. Nowadays, in some regions of Africa and Asia it is possible to find J. curcas. It is extremely resistant to long dry conditions. After 120 hours of fermentation with A. terreus, 24.45 g/l of ITAC were produced. El-Imam et al., (2013) used JSC to produce ITAC by A. terreus but tried different optimized medium and conditions from the work done in 2007. Maximum yield of ITAC was 48.70 g/l (submerged fermentation) in the following conditions: 5 ml of inoculum size in total volume of 50 mL, 50% substrate concentration and pH 1.5. Despite that, the authors report as optimized physic-chemical parameters for fermentation of JSC to produce ITAC, the use of pH 4, inoculum size of 5 ml and JSC concentration of 40%.

Almost 60% of annual whey production worldwide is transformed into products such as whey proteins. However, the lactose-rich waste stream is still almost useless for other markets, which makes it a waste. The application of this waste would help to solve the problem of the high pollution potential associate with disposal of this stream (Panesar & Kennedy, 2012). Considering its wide availability, low cost and non-competitiveness with food sources, whey may provide not only a sustainable, but also a cost-effective feedstock for producing organic acids.

Omojasola & Adeniran (2014) used sweet potato peel (SPP) (an agro-based waste) for ITAC production by A. niger (ATCC 16404) and A. terreus (ATCC 20542). SPP has an interesting composition: 65.9 % on carbohydrate; 22.60% on sugars; 5.38 % protein. In A. niger, 67.67 g/l of ITAC were obtained and in A. terreus 70.67 g/l from this substrate. As expected, after optimization of the conditions, i.e., when all the best conditions tested for each parameter, pH, substrate concentration, temperature and inoculum size were put together, higher results were obtained at values of 112.67 g/l for A. niger and 115.67 g/l after five days of A. terreus fermentation at pH 4.0, 10% substrate concentration and 5 mL inoculum size.

2.7 Downstream strategies Downstream methods represent between 50 to 70% of the total costs in organic acids production (Cheng et al. 2012). Specifically, considering carboxylic acids, Straathof (Straathof 2011) states that downstream represents 30 to 40% of the total production costs. During the last years, several methodologies were used for ITAC separation from the fermentation broth. One of the most important factors was the discovery that in ITAC concentrations above 25 g/l, this acid can have interference in its own production (Klement et al, 2012), and that is a reason why ITAC titres did not reach yet the theoretical titre. In its extensive review in 2014 for organic acids downstream, López-Garzón defines 6 important points in downstream processing: clarification, primary recovery, counter ion removal, concentration/purification, upgrading and formulation (López- Garzón & Straathof 2014).

Cell removal is usually the first downstream process step for extracellular products and it is done by filtration or centrifugation in the end of the fermentation, when production is made in batch. When continuous fermentation is performed, the common process is to immobilize the microorganisms, which depending on immobilization type can have different procedures. Different methods have been applied for ITAC downstream along the years and Magalhães AI Jr. et al. (2016) summarized quite well these studies (Table 3).

Table 3-Different downstream methods applied during the years (Adapted from (Magalhães AI Jr. et al., 2016))

Method ITAC solution Yield (%) Reference Crystallization Fermentation broth 51 Dwiarti et al. (2007) Crystallization Aqueous solution 83 Hogle et al. (2002) Crystallization Fermentation broth 81 Lockwood & Ward (1945) Crystallization Fermentation broth 80 Okabe et al. (2009) Precipitation Fermentation broth 51 Kobayashi & Nakamura (1971) Reactive Extraction Aqueous solution 98 Kaur & Elst (2014) Reactive Extraction Aqueous solution 73 Wasewar et al. (2011) Electrodialysis Aqueous solution 98 Fidaleo & Moresi (2010) Diafiltration Fermentation broth 60 Carstensen et al. (2013) Adsorption Aqueous solution 100 Magalhães Al Jr. (2015) Adsorption Aqueous solution 100 Schute et al. (2016)

Figure 3- Industrial downstream ITAC process (retrieved from Magalhães Al Jr et al (2016))

The industrial process for ITAC recovery is done with crystallization process. This process is described in the literature by several authors and by Magalhães Al Jr et al (2016).

This is an evaporation-crystallization process where water is evaporated to concentrate ITAC, but this process is not able to separate some of by-products of the fermentation, especially if they are also organic acids of similar solubility, results in low purity of ITAC. One of the first known ITAC recovery methods present in the literature was developed by Lockwood & Ward. From the fermentation broth of A. terreus NRRL 1960, the authors say that approximately 80% of the ITAC is present in crystals, while 20% stay in the fermentation broth.

In 1971, Kobayashi & Nakamura (1971) suggested a process for recovery of ITAC from fermentation broth by adding basic lead carbonate and increase the temperature to 100 ºC to precipitate the formed salt.

In 2002, Hogle et al. tried to recover ITAC from synthetic aqueous solutions of citraconic and succinic acid by crystallization. In this work, succinic acid was first selectively crystallized since it has low solubility and ITAC was crystallized after. In a second stage, 72% of ITAC was recovered with 98.5 % of purity.

Dwiarti et al (2007) recovered from A. terreus TN484-M1 with sago starch or glucose as carbon source, 51.3 % of ITAC with 97.2% of purity. With the glucose fermentation, 51.4% of ITAC was recovered with 99% of purity. The process used the steps of filtration, evaporation, crystallization, re-crystallization and drying. The purity of ITAC made differences in the melting point of the ITAC recovered from both fermentation broths. Okabe et al. (2009) hypothesised a process using crystallization for ITAC recovery with 80% of yield. Crystallization is a method with the advantage of high purity and efficiency, but the overall cost and energy of the process are clearly bottlenecks for its use.

Electrodialysis was used by Kobayashi et al. (1975). Application of an electric field promote charged species to migrate and separate from the uncharged particles. Fidaleo & Moresi (2010) apply the Nernst–Planck equation to model the recovery of ITAC through electrodialysis with univalent electrolytes; the model considers the ITAC dianion feature, converting ITAC into a

17 disodium salt. About 98% of ITAC from the aqueous solution was recovered by this method. As referred before, Carstensen et al. (2013) used a continuous bioreactor with reverse-flow diafiltration (RFD). This system was developed by the same group in 2012, where the product stream goes through a hydrophilic ultrafiltration hollow-fiber membrane that is placed in the bioreactor and where with defined time-points the flow is reversed. The result with theoretical pure ITAC showed 100% of recover while with the fermentation broth of U.maydis fermentation, only 60% of ITAC was recovered.

Many tests were done for liquid extraction with organic solvents for ITAC recovery. However, organic acids are more soluble in water than in any of the organic solvents (alcohols, esters, ethers), which make more difficult its separation in these solvents because of the low distribution coefficient. To overcome this problem, a new technique was used, called reactive extraction (RE). In this process, an extractant (tertiary or quaternary amine) is used. The ITAC originally in the aqueous solution is extracted to the organic phase with the reactive extractant. The acid and extractant make a complex that has affinity for the organic phase. The reaction is then reverted by mixing with the acid phase to where the organic acid is recovered, while organic phase and extractant are recycled.

Methods of separation of ITAC using adsorbents have been applied during the last years. In this process, an absorbent (solid platform with affinity to the desire compound to separate) is used to select the product of interest by affinity. The goal is to let ITAC attached to this adsorbent while all the fermentation broth components flow through the same absorbent. The elution of ITAC can then be done with another strategy. An example of an adsorbent are the ion-exchange resins. ITAC can vary between 3 different protonation states. In two of them, the charge of ITAC is negative as a mono or divalent anion. Taking advantage of the properties of the adsorbents, an anion-exchange resin would be theoretically working for ITAC separation from the uncharged components of the fermentation broth. Magalhães Al Jr. et al. (2016) tested two commercial strongly basic ion-exchange resins for the recovery of ITAC. PFA-300 achieved a recovery yield of 0.23 g/g dry resin at pH 3.85. Schute et al. studies the hypothesis of separate ITAC by a liquid phase adsorption on highly hydrophobic adsorbents. These authors synthesized and used Hyper- Cross-Linked Polymer (HCP), activated carbon, among others. The best result was achieved with activated carbon, NORIT® A Supra EUR with 0.42 g of ITAC per g of activated carbon.

In the elution phase, or by other words, the step where ITAC was recovered from the adsorbent, the choose of the eluent is important depending of the characteristics of the acid in the adsorbent. When a neutral absorbent is used, ITAC binds to it on its neutral form, the use of an eluent able to shift ITAC for its anionic form can disrupt the connection of ITAC with the adsorbent. However, when the acid is bind to the resins in the deprotonated form, then, conversion to its neutral form to detach the ITAC from the adsorbent. Another important characteristic is that the use of an eluent may not destroy the adsorbent, allowing to recycle it into another separation process, and that decrease production costs.

3. Materials and Methods

3.1 Materials Reagents: D-glucose (Fischer Chemicals); ammonium sulphate (PanReac AppliChem); Yeast Nitrogen Base (YNB) from Difco; potassium phosphate (Merck); cheese whey (kindly provided by Lacticínios do Paiva, S.A in Lamego, Portugal) as Miguel Nascimento’s connection; sulphuric acid 96% (Fischer Chemicals); ITAC > 99% (Sigma-Aldrich), Citric acid > 99% (Sigma-Aldrich), acetic acid (Fischer Chemicals)

Autoclave: Uniclave 88 - AJC

Centrifuges: Eppendorf centrifuge 5810R, Rotor FA-45-6-30 (centrifuge 1); Sigma, Sartorius 1- 15P (centrifuge 2)

Incubator: Aralab, Agitorb 200 (incubator 1); Memmert (incubator 2)

Bioreactors: FerMac 360 from Electrolab Biotech Limited, 2 L vessel

Spectrophotometer: HITACHI U-2000

Filters: 47 mm Hydrophilic Nylon Membrane Filter with a 0.2 µm pore size, MF-Millipore™

3.2 Strains used and maintenance Saccharomyces cerevisiae (BY4741) and Issatchenkia orientalis (SD108) strains (uracil auxotroph) were used. Both contain a plasmid containing the coding sequence of the A. terreus enzyme CAD (AtCAD), allowing the synthesis of ITAC. For S. cerevisiae BY4741, plasmid contains a strong galactose promoter (pGal1-AtCad1). For I. orientalis, it contains a glucose- inducible promoter. Both strains were kindly provided by iBB-IST (Prof. Nuno Mira’s group). Both strains were maintained in MMB (Minimal Growth Medium) Ura- solid growth medium, containing, per liter, 20 g glucose, 1.7 g Yeast Nitrogen Base (without amino acids and ammonium sulphate) and 2.65 g ammonium sulphate (Merck). Cultures were grown at 30º overnight (incubator 2) and then kept at 4ºC and periodically renewed by cultivation of cells from a previous MMB to new MMB. The growth of this renewed culture in MMB was done in incubator (incubator 2) at 37ºC overnight and kept at 4ºC after that.

3.3 Culture Medium and cultivation conditions Cultivation in liquid medium for fermentation assays (pre-inoculum) was performed in MMF (Minimal Fermentation Medium) medium, with 20 g/l glucose, 2.65 g/l ammonium sulphate, 1,7 g/l Yeast Nitrogen Base (YNB), 2 g/l of potassium phosphate and amino acids, vitamins and trace elements denominated as Complete Supplement Mixture (CSM – in appendix) in 50 mL of volume in a 250-mL shake-flask, and incubated (incubator 1) overnight at 250 rpm and 30 ºC. MMF medium was sterilized in the following way - for 1 L preparation: 750 ml of mili-Q water were added to the medium components and autoclaved at 120 ºC and 1 bar for 20 minutes. 250 mL of

CSM were sterilized using a sterilized filtration system since the amino acids may be temperature sensitive. After this procedure, CSM is added to the autoclaved medium and trace elements and vitamins are added, in the proportion of 1 mL to 1L of MMF.

3.4 Biomass determination and sugar and metabolites quantification Culture samples of 1 mL were collected and the OD at 600 nm was measured to evaluate the cell growth and quantify biomass. For I. orientalis cultures, 1:200 dilution was done to make the values of absorbance (Abs) in the range of the calibration.

Another mL of the sample was taken and centrifuged for 5 minutes at 10 000 rpm (centrifuge 2). Supernatants were collected, filtered with 0.2 µm filters and analyzed for galactose, glucose, lactose, ITAC and other fermentation by-products quantification by High Performance Liquid Chromatography (HPLC). HPLC analysis was then performed using as mobile phase a sulfuric acid solution (0.005M) and injecting 10 µL of this supernatant in a Bio-RAD Aminex HPX-87H® column with a flow rate of 0,6 mL/min. UV detector at 210 nm coupled to the HPLC was used for the detection of organic acids and a RI detector for the detection of sugars, ethanol, and glycerol.

3.5 ITAC production with batch fermentations of I.orientalis and S. cerevisiae in shake- flasks S. cerevisiae BY4741 and I. orientalis SD108 were cultivated for pre-inoculum as described before. In the next day, to the fermentation step, the OD of the pre-inoculum was measured at 600 nm and an inoculum volume was selected to reach an OD of 0.1 in the beginning of fermentation. This volume was then centrifuged and cells collected and transferred to different 250 mL shake-flasks, containing different 50 mL MMF medium with 20 g/l galactose (for S. cerevisiae or glucose for I. orientalis) and assuring a 1:5 medium/air ratio during the fermentation. Cultures were maintained at 250 rpm and 30º, and 2 mL daily samples were taken to measure the growth of the cultures by OD (600 nm) and the presence of extracellular metabolites, in particular ITAC, by HPLC (High Performance Liquid Chromatography). S. cerevisiae cultures were carried out for 10 days and I. orientalis from 5 to 8 days.

3.6 ITAC production with feed-batch fermentation of I. orientalis in shake-flasks I. orientalis was cultivated for pre-inoculum as described before. In the next day, pre-inoculum volume was selected, centrifuged and the cells were collected and transferred to a new 250 ml shake-flask with 50 mL of new MMF medium for the OD at the beginning of fermentation to be 0.1. Conditions were assessed:

• Condition A (control): MMF medium (20 g/l of glucose, 2.65 g/l of ammonium sulphate, 1.7 g/l of YNB and 2 g/l of potassium phosphate + CSM). No further substrate addition was done. • Condition B: MMF medium (40 g/l of glucose, 2.65 g/l of ammonium sulphate, 1.7 g/l of YNB and 2 g/l of potassium phosphate + CSM). No further substrate addition was done.

• Condition C: Initial fermentation medium equal to control. An addition in the first (24h) day of fermentation to the shake-flask of 2.5 mL of a solution of 400 g/l of glucose. (Implying an addition of 20 g of glucose per liter of fermentation broth) • Condition D: Initial fermentation medium equal to control. An addition in the second (48h) day of fermentation to the shake-flask of 2.5 mL of a solution of 400 g/l of glucose. (Implying an addition of 20 g of glucose per liter of fermentation broth) • Condition E: Initial fermentation medium equal to control. Daily feeds of 2.5 ml from a solution of 400 g/l of glucose were done (20 g of glucose per liter extra feed) • Condition F: Initial fermentation medium equal to control. Daily substitution of 12.5 mL of the total volume of the culture for new 12.5 mL of new fresh MMF medium (four times concentrated) was done. The 12.5 mL of taken medium were centrifuged and the cells were put back into the culture to maintain the biomass.

Daily samples were collected and analyzed for cell growth (spectrophotometry) and extracellular metabolites (HPLC)

3.7 Study of the toxicity of the cheese whey in I.orientalis

Cheese whey: Pre-inoculum was done as before. Cheese whey was clarified and sterilized. Using a 3-step protocol that includes its filtration (with 0.2 µm flter) followed by the immersion of the falcons with whey during 15 minutes at 90 ºC to denature the protein content; a centrifugation (centrifuge 1) at 10 000 g for 10 minutes to remove these components and collection of the supernatant in sterile shake-flasks, comprising the clarified whey to be used on the fermentations.

An inoculum volume was selected to reach an OD of 0.1 in the beginning of fermentation. This volume was then centrifugated and cells collected and transferred to 50 mL of clarified cheese whey in a 250-mL shake-flask. During the five days of fermentation, samples were taken and analyzed for cell growth (spectrophotometry) and extracellular metabolites (HPLC).

Cheese whey and MMF medium: Five conditions were assessed:

• Condition A (control): MMF medium (20 g/l of glucose, 2.65 g/l of ammonium sulphate, 1.7 g/l of YNB and 2 g/l of potassium phosphate + CSM) • Condition G: MMF medium was added in 50% v/v and clarified cheese whey was added in the other 50% v/v. The final concentration was 20 g/l of glucose, 2.65 g/l of ammonium sulphate, 1.7 g/l of YNB and 2 g/l of potassium phosphate. • Condition H: MMF medium was added in 95% v/v and clarified cheese whey was added to 5% v/v. The final concentration was 20 g/l of glucose, 2.65 g/l of ammonium sulphate, 1.7 g/l of YNB and 2 g/l of potassium phosphate.

• Condition I: MMF medium was added in 99.5% v/v and 0.5% v/v of clarified cheese whey was added. The final concentration was 20 g/l of glucose, 2.65 g/l of ammonium sulphate, 1.7 g/l of YNB and 2 g/l of potassium phosphate.

• Condition K: Initial fermentation medium equal to condition G. Daily feeds of 2.5 ml from a solution of 400 g/l of glucose were done (20 g of glucose per liter extra feed)

Elucidation of additional HPLC Peak

I. orientalis with the plasmid containing the AtCAD gene and I. orientalis with no plasmid inserted (empty) were cultivated in MMF medium (added in 50% v/v and clarified cheese whey was added in the other 50% v/v. The final concentration was 20 g/l of glucose, 2.65 g/l of ammonium sulphate, 1.7 g/l of YNB and 2 g/l of potassium phosphate. Cultures were incubated (incubator 1) during 5 days at 30 ºC and 250 rpm. Cell growth was measured to assure that the fermentation was regular, and extracellular metabolites were measured by HPLC.

Cheese whey hydrolyzation

Cheese whey was hydrolysate using HCl 10M. As an example, 200 mL of clarified cheese whey containing 105 g/L of lactose were heated up to 90 ºC and 45 mL of HCl 10M was added to start the hydrolysis reaction for 1 hour. The pH of the hydrolyzed cheese whey is too acidic to be used in fermentation and was then adjusted to pH 4 with NaOH 5M.

3.8 Bioreactor fermentations with synthetic medium (conditions L, M, N, O) Assessment of ITAC production in bioreactors was performed using the same pre-culture procedures for I. orientalis. For 1 L fermentation, 750 mL of MMF medium was autoclaved inside the bioreactor. CSM was sterilized using filtration in a 0.2 µm filter and vitamins and trace elements were added (relation of 1 mL per liter of medium). The OD of the pre-inoculum was measured, and calculations were made use a pre-inoculum that ensures an initial OD of 0.1. This volume was then centrifuged to separate the pre-inoculum medium from the cells that will start the fermentation process. The pellet was then resuspended with a volume of CSM with vitamins and trace elements, and this mixture was then placed into the bioreactor. Dissolved oxygen (set point 15) was controlled with the air flow (automatic between 0 and 1 vvm) and agitation (between 150 and 400 rpm) in a cascade loop control. Temperature was set for 30º C and maintained with a heating jacket and cold water in circulation. pH and DO sensors were previously calibrated. pH was controlled (set point 4) in one of the reactors with KOH 0.5 M. Daily samples of about 5-7 ml were taken for OD measure and extracellular metabolites monitoring. In the bioreactors with daily glucose feed, 50 mL of a 400 g/l glucose solution were added to the bioreactor to perform a 20 g/l addition of glucose.

3.9 Bioreactor fermentations with cheese whey (conditions P, Q) Assessment of ITAC production in bioreactors was performed using the same pre-culture procedures for synthetic medium. For 1 L fermentation, 375 mL of MMF medium was autoclaved inside the bioreactor. CSM was sterilized using filtration in a 0.2 µm filter and vitamins and trace elements were added (relation of 1 mL per liter of medium). To this CSM, 500 mL of sterilized cheese whey were added, making a total of 625 mL. The OD of the pre- inoculum was measured, and a volume of pre-inoculum was added to ensure an OD of 0.1 at the beginning of fermentation. This volume was then centrifuged to separate the pre-inoculum medium from the cells that will start the fermentation process. The pellet was then resuspended with a volume of CSM with vitamins and trace elements and clarified cheese whey, and this mixture was then placed into the bioreactor. Dissolved oxygen (set point 15) was controlled with the air flow (automatic between 0 and 1 vvm) and agitation (between 150 and 400 rpm) in a cascade loop control. Temperature was set for 30º C and maintained with a heating jacket and cold water in circulation. pH and DO sensors were previously calibrated. pH was controlled (set point 4) in one of the reactors with KOH 0.5 M. Daily samples of about 5-7 ml were taken for OD measure and extracellular metabolites monitoring. In the bioreactors with daily glucose feed, 50 mL of a 400 g/l glucose solution were added to the bioreactor to perform a 20 g/l addition of glucose

3.10 Use of polybenzimidazole (PBI) as an adsorption polymer for ITAC and citric acid separation PBI was made in iBB-IST and used as an adsorption polymer for separation of ITAC and citric acid. 50 mg of PBI was used for 1 mL of solution. Solutions of ITAC and citric acid were prepared, with 20 g/l of ITAC and 40 g/l of citric acid. pH of the solutions was adjusted for 3.5, 4.7 and 7, or maintained as original, 1.8. During 24h, the solution and PBI were in contact and a magnetic agitator was used to better mix. After this 24h, the magnetic agitator was removed and the solution and PBI were centrifuged (centrifuge 2). The supernatant was collected and analyzed by HPLC.

4. Results and discussion 4.1 ITAC production with batch fermentations of I.orientalis and S. cerevisiae in shake-flasks The production of ITAC with both strains was evaluated in shake flask in the first part of this study. Both strains were already tested for ITAC production before in iBB, in previous studies. Here, an initial comparison is performed to select the strain capable of potentially increase ITAC production by testing different cultivation conditions.

5 600nm

0.5 0 1 2 3 4 5

0.05 Fermentation time (days)

Figure 4-Cell growth in S. cerevisiae BY4741 (blue squares) and I.orientalis SD108

(orange circles)

30 1

(g/l) (g/l)

20 )

0.5 g/l

acetic acetic acid 0.5

and citric acid ITAC ( ITAC 10 (g/l) ITAC

Glucose Glucose Galactose and and Galactose 0 0 0 0 0 1 2 3 4 5 0 1 2 3 4 5 Fermentation time (days) Fermentation time (days) Figure 5-Fermentation broth analysis for: a) I. orientalis SD108 – Glucose consumption (grey squares), ITAC (blue triangles) and citric acid (orange circles) production; b) S. cerevisiae BY4741– Galactose consumption (grey squares), ITAC (blue triangles) and acetic acid (green diamonds).

The cell growth curve for each species is very different, with higher capability of I.orientalis to divide and multiply than S. cerevisiae BY4741 (Figure 4). Figure 5 shows significantly differences between the two-species concerning the quantity of ITAC produced, the rate of carbon source consumed, and the by-products formed. In the case of S. cerevisiae, at the day five during fermentation, approximately 33 mg/l of ITAC were produced and the main by-product of the

24 fermentation is acetic acid. The best ITAC result obtained in iBB for S. cerevisiae BY4741 is 65 mg/ml after 14 days of fermentation (Santos, 2016). In contrast, in I. orientalis, at the same day of fermentation, approximately 500 mg/l were already produced while in this case the main by- product of the fermentation is citric acid. In both cases, and in these conditions, the by-product has a higher concentration than the product of interest. Another different characteristic between the species is their ability to metabolize sugars. Both species were tested in medium with galactose or glucose. In the case of I. orientalis, the growth was only noticed when glucose was in the medium while when the medium has galactose, no growth was observed (data not shown). In the case of S. cerevisiae, both sugars are metabolized.

According to the best ITAC producer, I. orientalis, the process upgrade and scale-up was made with this specie only. Two objectives were clearly defined, the first one to obtain the best titre, carbon yield and productivity for ITAC production and as important as this one, to reduce the by- product formation and avoid new by-products. Looking at the metabolic point of the process, citric acid is a precursor of ITAC, which states the importance of produce first citric acid that needs to be converted to ITAC. However, high amounts of citric acid by the end of the fermentation is not an advantage for the process. The values for ITAC production with I. orientalis are comparable with the results obtained with other engineered microorganisms with the same initial sugar source, as example is Blazeck et al. (2015) with Y.lipolytica, obtaining 160 mg/l with optimized conditions in shake-flask. The value obtained in this work is 3-fold higher than the value obtained in Y.lipolytica.

4.2 Strategies to improve ITAC and by-product decrease production in synthetic medium with I.orientalis

4.2.1 Effect of carbon source feed regime and initial concentration on ITAC production One of the purposes of this work is to assess if a different initial concentration of carbon source can influence biomass and ITAC/citric acid production in I.orientalis. The cell can use the carbon source for growth, survival, as example the cell redox potential maintenance, and organic acids production. Therefore, the carbon source supplied that is used for ITAC production is limited. To understand in which way I. orientalis culture is affected by different carbon source feeding approaches and understand how can this carbon yield to produce ITAC be optimized, four conditions were tested. Two batch fermentations were started with 20 and 40 g/l of glucose with no feed during the five days of fermentation (Conditions A and B). At the same time, two batch fermentations were started with 20 g/l of glucose, but another 20 g/l were added at first (Condition C) or second day (Condition D) of fermentation time. Results for optical density, ITAC and citric acid production and carbon source consumed are displayed in Figure 6.

100 1 40 2

30 10

600nm 20 OD

1 (g/l) Glucose 0 1 2 3 4 5 10

0 0.1 Fermentation time (days) 0 1 2 3 4 5 Fermentation time (days)

2.5 1.5 3 4

2.0 1.0 1.5

1.0 ITAC (g/l) ITAC

0.5 (g/l) acid Citric 0.5

0.0 0.0 0 1 2 3 4 5 0 1 2 3 4 5 Fermentation time (days) Fermentation time (days)

Figure 6- Shake-flask fermentations with I. orientalis using different initial sugar concentrations and feed strategies (conditions A to D). Condition A – Blue circles; B- purple squares; C – red triangles; D- Green diamonds; 1- Biomass growth in optical density; 2- Glucose consumption; 3- ITAC production; 4- Citric acid production.

The cell growth was measured using spectrophotometry, and the values are displayed in optical density (600 nm). The growth of the conditions A, C and D is very similar during all the fermentation time. However, in the condition B, when the initial concentration of glucose is 40 g/l, the biomass produced is almost the double of the biomass produced by the other three conditions. Comparison of the ITAC production in conditions A and B shows that in the condition B the production is slower but in the end equivalent to the condition A. Therefore, the higher initial glucose concentration in the condition B, did not lead to higher ITAC production in the first five days of fermentation. This suggests that the cell prefers to use the initial sugar source for biomass production and additional carbon source supplied is not used to produce organic acids. The use of additional glucose feeds (conditions C or D) was compared with the control (condition A) lead to almost the double of ITAC production. Therefore, the use of higher amounts of carbon are more efficient for ITAC production if divided between two time-points, instead of adding more carbon source in the beginning if the aim is to produce ITAC. In the condition B, at the fifth day of fermentation, 0.64 g/l of ITAC was produced. In the conditions C and D, 1.13 g/l and 0.98 g/l respectively were produced. However, it is important to refer that the best carbon yield was

26 obtained for the condition A (Table 4). This result shows that a higher initial sugar source concentration is not directly related with higher ITAC production but rather with biomass production. It is like building the factory (producing biomass) and then start to operate on the production (ITAC production) after the addition of sugar source after the cell growth.

Regarding the by-products of fermentation, and as before, the main and only by-product detected in I. orientalis culture fermentation broth was citric acid. In the conditions A and B, the concentration of citric acid is approximately constant during the fermentation time. However, In the condition B after the third day of fermentation, a slightly increase in citric acid concentration was observed. In the conditions C and D, where the glucose feed was done, the levels of citric acid in the extracellular medium decrease during the fermentation time. The glucose feeding seems to have contribution in the decrease of citric acid concentration over the fermentation broth. A possibility for this is that citric acid is being converted to ITAC, which would explain the increase in ITAC and decrease in citric acid when compared with the control. The mechanism through this possible phenomenon is promoted by the addition of glucose is not elucidated in this experiment. The uptake by the cell of citric acid is a possibility since the cell would lack carbon source for its activity and so, uses citric acid back in TCA cycle, which can be related with the increasing of ITAC over the fermentation time.

With the results obtained, there is room to think about a fed-batch or even continuous fermentation process for ITAC production. So, the next conditions tested is the daily feed or the carbon source, in the case glucose, on the concentration of 20 g/l. Also, was tested the possibility of adding not only carbon source, but the addition of the medium MMF daily and check if the medium components more than only the carbon source make difference on ITAC production.

Table 4- Summary of the ITAC and citric acid titres in the different fermentation conditions with I. orientalis. Productivity and yield were calculated for ITAC Condition Final ITAC Final Citric ITAC yield from Productivity (g.l-1.h-1)

(g/l) (g/l) glucose Condition A 0.67 1.53 0.033 0.006 Condition B 0.64 0.85 0.016 0.005 Condition C 1.13 0.17 0.028 0.009 Condition D 0.98 0.17 0.025 0.008

4.2.2 Daily sugar source and medium feed towards a fed-batch or medium renew fermentation Another test in the process and regarding the previous result was to understand if a daily carbon source feed would favor higher titres of ITAC. At the same time, instead of the carbon source only, the change of the fermentation medium could also be benefic for the increasing of these titres. So, daily, 20 g of glucose per liter was added to the fermentation broth (Condition E) and in the other condition (Condition F), 25% of the volume of MMF medium was substituted in the culture by a renewed four times more concentrated MMF medium, leading to a final concentration

of 20 g/l of glucose daily, but this time all the MMF medium components were added also, like nitrogen source or amino acids. Summary of the results obtained is in Figure 7.

100 1 20 2

15 10

600nm OD 1 (g/L) Glucose 5 0 1 2 3 4 5

0 0.1 0 1 2 3 4 5 6 7 8 Fermentation time (days) Fermentation time (days) 3.0 3 4 4 2.5 3

2.0

1.5 2 ITAC (g/l) ITAC 1.0 citric acid (g/l) acid citric 1 0.5

0.0 0 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 Fermentation time (days) Fermentation time (days)

Figure 7- Shake-flask fermentations with I. orientalis using different initial sugar concentrations and feed strategies Condition E – blue squares; Condition F – orange circles; 1- Biomass growth in optical density; 2- Glucose consumption; 3- ITAC production; 4- Citric acid production. For glucose consumption, the glucose added daily was not measured and it is considered the theoretical value of glucose upon addition. The values for time zero and 24h after every glucose addition were measured.

In the Condition E, the behavior of the culture in the first three/four days of fermentation exhibits the same behavior of previous fermentations (conditions A to D), but after this day, the culture

presented a slightly green color relatively to the control (condition A). A decreasing in the rate of

ITAC production levels is observable during the fermentation time. To mislead contaminations, a sample from the culture was taken and cultivated in MMB Ura- medium at day five of fermentation (Figure 8). The medium shows that only I. orientalis is present. The cultivation of the cells (Figure 8) shows also that the cells did not suffer lysis and so, this different color of the culture is not due to the disruption of the cells and the unification of intra and extracellular medium. Since glucose has being added to the fermentation broth, one possibility is that the cells are entering in stress, maybe because they have glucose available but still they have not the other needed nutrients per example to multiply and grow, and so, they react by producing some compound that is not detectable in HPLC, like per example a carotenoid. Other theory relates with the reactive oxygen species (ROS) production. ROS are formed during the normal metabolism in mitochondria as a

28 derivation of molecular oxygen, but when they are present in excess, per example under stress conditions, it is known that in yeasts, carotenoids are produced with the primary function of fight this ROS. An example of carotenoid production in yeast is Phaffia rhodozyma, where under ROS stress, carotenoids where produced and act as antioxidants. Schroeder & Johnson (1995) suggest that ROS may activate carotenoids synthesis by gene activation.

a) b)

Figure 8-- a) MMB Ura- medium cultivated with fermentation medium from condition E; b) Shake-flasks of fermentation in condition E with I. orientalis

In the Condition F, 25% of the medium was taken out and substituted by the same amount of new medium, but four times more concentrated to make the final concentrations of carbon source and medium components as the beginning of the fermentation. This test led progressively to higher values of biomass, with the cell growth being visible daily. However, the amount of ITAC produced (Figure 7) is not higher than the one produced in Condition E. The daily glucose feed seems to have an improved impact on ITAC production that the medium substitution, and so, a better strategy to follow when scaling-up the process. Both conditions produced higher ITAC titres and obtained higher productivity than the control (Condition A). However, the carbon yield is lower in both conditions.

4.3 Strategies to produce ITAC using cheese whey and hydrolysate as culture supplement or alternative substrate

4.3.1 Study of the toxicity of the cheese whey in I. orientalis and S. cerevisiae

As mentioned, the use of cheaper substrates rich in carbon source is of special interest regarding the economy of the process. Cheese whey is an alternative substrate for ITAC production regarding the fact that is cheap and has around 4 to 5% of its volume in weight of lactose. The first step of this study is to understand if cheese whey is toxic for the cells and can be used as substrate. For this, cheese whey was tested directly in I. orientalis and S. cerevisiae for the assessment of cell growth and ITAC production. Both species were not able to grow in the shake- flask fermentations in the same conditions as the synthetic medium (data not shown). The analysis by HPLC revealed that the sugar present in the cheese whey, lactose (a disaccharide of two monosaccharides, glucose and galactose) is not consumed in any of the fermentation days, which reveals that these two species are not able to metabolize the lactose naturally. This result agrees with the literature where wild-type S. cerevisiae was known to not metabolize lactose (Guimarães et al. 2010). So, two ways can be explored: to use cheese whey as a sugar source after a treatment to break the lactose and make glucose and galactose available in the medium or; to use cheese whey as a rich-medium supplement for the fermentation. To understand the toxicity/benefits of the cheese whey to the cells regarding this second option, different clarified cheese whey concentrations were tested to evaluate toxicity and possible differences in ITAC production during the fermentation time. Condition A (control), Condition G (50 % v/v of cheese whey and MMF medium), Condition H (5 % v/v of cheese whey and 95% v/v MMF medium) and Condition I (0.5 % v/v cheese whey and 99.5 % v/v MMF medium) were tested. a) 10 b)

600nm 1

OD 0 1 2 3 4 5 6 7 8 9 10

0.1

Ferrmentation time (days) Fermentation time (days)

Figure 9- Shake-flask fermentations with a) - I. orientalis and b) - S. cerevisiae with different concentrations of cheese whey in the medium (conditions A, G, H and I); Condition A (orange dots); Condition G (blue circles); Condition H (grey triangles) and Condition J I (yellow squares). Biomass growth in optical density.

30 2.5

20 1.5

1 (g/l) ITAC

(g/l) Glucose 10

0.5

0 0 0 1 2 3 4 5 Fermentation time (days)

Figure 10- Shake-flask fermentations with I.orientalis with different concentrations of cheese whey in the medium (conditions A, G, H and I); ITAC concentration and glucose consumption during the fermentation time– Condition A (orange diamonds), Condition G (blue circles), Condition H (grey triangles), condition I (yellow circles).

Comparison of the control (Condition A) with any of the different tested conditions shows that cheese whey is not toxic for both I.orientalis and S. cerevisiae.

The cell growth has approximately the same behavior in all the conditions (Figure 9), with the growth in the first day (where the carbon source was used, among other, to cell division) and the stationary phase in the subsequent days. This non-inhibition of growth can lead this work further regarding the possibility of adding cheese whey as a rich medium supplement in the process of ITAC production (Figure 10).

Table 5-Summary of the ITAC and citric acid titres obtained in conditions A, G, H and I. Productivity and yield were calculated for ITAC Condition ITAC (g/l) Citric acid ITAC yield from Productivity (g.l-1.h-1) (g/l) carbon Condition A 0.67 1.53 0.033 0.006 Condition G 1.99 4.75 0.095 0.016 Condition H 0.76 4.07 0.038 0.006 Condition I 0.6 2.78 0.030 0.005

Citric acid (g/l) acid Citric 2

0 0 1 2 3 4 5

Fermentation time (days)

Figure 11- Shake-flask fermentations with I.orientalis with different concentrations of cheese whey in the medium (conditions A, G, H and I); Citric acid concentration during the fermentation time in conditions A, G, H and I; Condition A (orange diamonds), Condition G (blue circles) , Condition H (grey triangles), condition I (yellow circles).

The only by-product with significant concentration in the medium in cheese whey fermentation is citric acid. The profile of citric acid in the extracellular medium is similar in tested conditions A, H and I (Figure 11). After the first 24 hours of fermentation, between 1.5 and 2 g/l of citric acid are formed and excreted to extracellular medium. For the condition H, where 50% v/v of cheese whey is used, the citric acid levels are around 4 g/l in the first 24 hours. After this moment, the concentration of this acid is almost the same until the end of fermentation, which means that the cell does not export this citric acid outside, using this acid internally or not-producing more citric acid since no more carbon source was added to the medium.

These results show that cheese whey can be used as supplement of the fermentation medium since the growth and production are not affected in any of the tested conditions and ITAC is improved. However, the presence of citric acid as a by-product in fermentation medium is not yet the ideal if considering the downstream process. The enhancing of the ITAC titre with supplement requires further elucidation. The best productivity and yield was obtained in this study under 50% v/v of cheese whey in the fermentation medium.

4.3.1.2 Elucidation of additional HPLC Peak

The HPLC analysis of the fermentation medium in cheese whey showed at the time zero of fermentation, peaks in the exactly retention time where ITAC and citric acid should appear on the chromatogram. The peak at retention time of citric acid is supported by the literature (Domingues et al., 2010) where citric acid, lactic acid, urea, and ureic acid are present in relevant quantities in cheese whey. However, there is no information regarding ITAC. To make confirmation that the

peak increasing in that retention time over the fermentation time correspond to ITAC production, a simple test was performed. Since I. orientalis is not a natural producer of ITAC, and the gene that codifies for its production is in the plasmid, a possible strategy is to do the fermentation with the empty plasmid vs the fermentation with the plasmid with AtCAD in a culture with 50% v/v of cheese whey and 50% v/v of MMF medium. With this simple test, we are assuring that what its producing its ITAC and no other kind of metabolite that this yeast can produce.

The fermentations with the empty plasmid produced more citric acid and no ITAC during the fermentation period (Figure 12). In opposite, in the fermentation using I. orientalis with the plasmid containing AtCAD, the peak for ITAC increased during the fermentation time, leading to the normal process. This can confirm that the results show only ITAC production and no other kind of metabolite present in cheese whey.

2.5 20 b) a) 2 15

1.5 10

ITAC (g/l) ITAC 1 Citric acid (g/l) acid Citric 5 0.5

0 0 0 1 2 3 4 5 0 1 2 3 4 5 Fermentation time (days) Fermentation time (days) Figure 12 – ITAC (a) and citric acid (b) productions in fermentations of I. orientalis with the empty plasmid (orange) vs the fermentation with the plasmid with Atcad (blue) in a medium with 50% v/v of cheese whey and 50% v/v of MMF

4.3.1.3 Hydrolysis strategy to transform cheese whey in a sugar source to I.orientalis Since none of the sugar source in cheese whey is consumed (I. orientalis is not able to break lactose), it is needed to give to the cells the sugars after the breakdown and not directly as cheese whey. Breakdown tests for lactose in cheese whey were performed.

Table 6 – Concentrations of lactose, glucose, and galactose in cheese whey before and after hydrolysis with 10M HCl

Sugar Initial concentration (g/l) Final concentration Lost in side reactions

Lactose 106.80 1.40 6.51 Glucose 0 48.59

Galactose 0 50.30

The final pH of the hydrolyzed cheese whey was 1.8. pH was adjusted to pH 4 to start the fermentation.

30 1.5

) g/l 20 1.0

Glucose (g/l) Glucose 10 0.5

ITAC and citric acid ( acid citric and ITAC

0 0.0 0 1 2 3 4 5 Fermentation time (days)

Figure 13- Analysis of the fermentation of I. orientalis in hydrolysed cheese whey at pH 4. ITAC (blue triangles), citric acid (orange circles) and glucose consumption (grey squares)

The results in the Figure 13 are from the fermentation made with hydrolyzed cheese whey with the strategy used before. In this case, 27.18 g/l of initial carbo source, glucose, was in the medium. The titre of ITAC is similar in the first day if we compare with the synthetic medium, but during the fermentation time, the ITAC concentration is still slightly increasing, which does not happen with the fermentation in synthetic medium. The citric acid levels are lower than in the synthetic medium, which less than half of the concentration. The use of cheese whey as a carbon source for I. orientalis in the production of ITAC was successfully done, with a productivity of 0.01 g.l-1.h- 1 and a carbon yield of 0.06. (Van Huyn & Decleire, 1982) stated that the number of bioproducts to be obtained after the hydrolysis of lactose into glucose and galactose would increase significantly since the majority of microoganisms cannot use directly lactose as a carbon source. Here, the example that I.orientalis can use cheese whey as a medium for fermentation was achieved after the hydrolysis was performed. Roukas, T. & Kotzekidou, P. (1991) showed the production of lactic acid from deproteneized whey using Lactobacillus casei and Lactococcus lactis cells. Colomban et al. (1993) showed the production of propionic acid from whey permeate. Now, ITAC production from clarified and deproteneized cheese whey was shown with no addition of any synthetic medium components.

4.3.1.4 Daily glucose feed in synthetic and cheese whey medium In another test performed to understand the effect of cheese whey in the fermentation process, daily glucose feeds of 20 g per liter were added to 50% v/v MMF medium and 50% v/v cheese whey (condition K) in comparison with the previous Condition E.

25 12

10 20 Condition K

8 15

10 (g/l) ITAC Glucose (g/l) Glucose 4

5 2 Condition E

0 0 0 1 2 3 4 5 6 7 8 Fermentation time (days)

Figure 14- Shake-flask fermentations with I.orientalis in two different conditions, E and K. Glucose consumption (grey squares) and ITAC production (Condition E- blue circles; condition K – orange triangles). For glucose consumption, the glucose added daily was not measured and it is considered the theoretical value of glucose upon addition. The values for time zero and 24h after every glucose addition were measured.

Condition K 5

Citric acid (g/l) acid Citric 2 Condition E 1

0 0 1 2 3 4 5 6 7 8 Fermentation time (days)

Figure 15- Shake-flask fermentations with I.orientalis in two different conditions, E and K. Citric acid production (Condition E- blue circles; condition K – Orange triangles)

Figure 16 – pH evaluation of the fermentation with I.orientalis in the conditions K and E. Photo taken at day 4 of fermentation

Figures 14 and 15 show the difference between the two fermentation conditions. While in condition E, the ITAC concentration increases regularly until the fourth day of fermentation and then this increasing starts to slow, in the condition K, the concentration of ITAC is increasing daily on the same pattern. The behavior of citric acid profile during fermentation is similar in both conditions, however, the concentration of citric acid in the condition K is higher than in the condition E.

The pH of fermentation samples was assessed through pH test strips. The difference between the two conditions is that when the cheese whey is present in the medium, the value of the pH is during the fermentation time between 4 and 5 during all the days of fermentation. In the synthetic medium only, the pH is close to 2. However, what is behind this cheese whey effect is not elucidated in this work.

4.4 Bioreactors

4.4.1 ITAC production using bioreactors Regarding the results obtained in shake flask for the best conditions to produce ITAC, tests in bioreactor were performed. The simplest case of production of ITAC was tested (Condition L) and at the same time, a bioreactor with the same conditions but pH control at pH 4 with KOH 0.5 M was tested (Condition M). Fermentation with pH control has been tested during the years for a wide variety of microorganisms and products to improve the titre of the desire product. pH 4 was chosen for I. orientalis fermentation because it was already tested before in I. orientalis as the best pH for succinic acid production (Xiao et al., 2014) and industrially contributes to decrease the probability of contaminations and acidic stress during the fermentation time. For five days, ITAC production, cell growth and other metabolites were measured.

On the first day of the fermentation, the OD of the culture with pH control (Condition M) is approximately two-fold higher than in the bioreactor without pH control and the differences on the first day of fermentation for ITAC concentration are of about five-fold (Figures 17 and 18), which reveals that pH control has a significant role on a higher concentration of the product in the fermentation broth. This result agrees with the results published in literature where pH control has influence on the titre of ITAC (Hevekerl et al. 2014; Blazeck et al. 2015). This can be due to the increase of cells viability inside the bioreactor, with higher viable cells density leading to more ITAC produced. Another positive hypothesis concerns the increase of the transport of ITAC from cytosol to extracellular medium. 80 mL of KOH (0.04 mol of K and 0.04 mol of OH) were consumed during the 6 days of fermentation. At this pH, more of the ITAC is in the monovalent carboxylic state that in condition L

100 Condition M

Condition L 10

600nm 1

OD 0 1 2 3 4 5 6

0.1

0.01 Fermentation time (days) Figure 17-Cell growth of I.orientalis in bioreactor with (squares) and without (circless) pH control. The control was made at pH 4 with KOH 0.5M

20 1

Condition M

10 0.5

ITAC (g/l) Glucose Glucose (g/l)

Condition L

0 0 0 1 2 3 4 5 Fermentation time (days)

Figure 18- Bioreactor fermentation with I.orientalis in conditions L and M. ITAC production (condition L – orange cicles and condition M – blue triangles) and glucose consumption (grey squares)

Regarding citric acid production (Figure 19), in both bioreactors the concentration of this by- product is higher than the concentration of ITAC. However, when the pH is controlled, the concentration of citric acid is around three-fold lower than when the pH is not-controlled. In both cases, the levels of citric acid and ITAC remain constant after the first day of fermentation. The production of more citric acid in more acidic pH has support in the literature (Papanikolaou et al., 2002; Blazeck et al.,2015). Hevekerl et al. (2014) showed an increase in 68% of ITAC production when the pH was controlled to 3. Here, 262% of ITAC production increase was obtained in five days of fermentation. However, the titre obtained for shake-flask is around three-fold higher than the titre obtained in bioreactor without pH control.

3.5

3.0 Condition L 2.5

2.0

1.5

Citric acid(g/l) 1.0 Condition M

0.5

0.0 0 1 2 3 4 5 Fermentation time (days)

Figure 19- Bioreactor fermentation with I.orientalis in conditions L and M. Citric acid production in condition L – orange circles and condition M – blue triangles

Cell viability in both bioreactors was also accessed. The differences in viability are visible in the dilution 10-5 with more viable cells for the condition with pH control (Figure 23). Blazeck et al. (2015) with Y. lipolytica produced 1.2 g/l of ITAC in bioreactor at pH 3.5 and using 80 g/l glucose as initial carbon source, with a carbon yield of 0.015 g/g and a productivity of 0.011 g. l-1.h-1. In the present work, 0.63 g/l were obtained at pH 4 with a carbon yield of 0.031 g/g and a productivity of 0.005 g. l-1.h-1. The results are very similar for both species, with a higher carbon yield obtained with I.orientalis and a higher productivity obtained with Y .lipolytica. After the overexpression of a cytoplasmic aconitase, 4.6 g/l of ITAC were obtained with a carbon yield of 0.058 g/g and a productivity of 0.045 g. l-1.h-1 for Y. lipolytica (Blazeck et al. 2015). A similar study can be performed with I. orientalis to increase the titre, productivity and carbon yield for ITAC production.

1 2 v

Figure 20 – Cell viability test in conditions M (1) and L (2) for I. orientalis fermentation in bioreactor

4.4.1.1 Daily sugar source feed as a precursor of a possible fed-batch or continuous fermentation After the test with a batch bioreactor with controlled (condition O) or without (Condition N) pH control, the same conditions were tested but in a fed-batch with daily addition of 20 g of glucose per liter.

20 3.0 Condition O

2.5

2.0

10 1.5 ITAC ITAC (g/l)

Glucose (g/l) 1.0

0.5 Condition N 0 0.0

0 1 2 3 4 5 6 Fermentation time (days)

Figure 21- Bioreactor fermentation with I. orientalis in conditions N and O. Itaconic acid production in condition N (orange circles) and condition O (blue triangles). In both bioreactors, a 20 g/l daily feed of glucose was made. Glucose consumption and addition profile (grey squares). For glucose consumption, the glucose added daily was not measured and it is considered the theoretical value of glucose upon addition. The values for time zero and 24h after every glucose addition were measured.

After 24h of fermentation, a 20 g/l glucose feed was applied to both bioreactors, and in both cases, was observed an increase in ITAC production when compared with the conditions without carbon source additions (Figure 21). However, the rate of the production is different, with an increase in concentration of ITAC in the bioreactor when the pH is controlled of 340% and 200% in the non- controlled one. The difference in ITAC concentration at the end of the fermentation time is from 2.8 g/l (condition O) to 0.34 g/l (condition N) where the only difference between them is the pH control and these results suggest that the pH is fundamental for the ability of the cells to be kept alive and in good conditions for ITAC production in bioreactor environment and because of that, the quantity of biomass. This can open the door for a process with pH control and carbon source pulses, per example, controlling glucose levels within a certain threshold and controlled by the addition of glucose. Cell growth revealed that pH control (Condition O) is very important for the growth and survival of the cells. The pH curve of the non-controlled pH reactor (Condition N) shows that before the 24 hours or fermentation, the pH of the medium is around 2.5 (Figure 23), which seems to affect the cell behavior compared with the pH 4, where both growth and survival and ITAC production are higher.

30 4

3 20 Condition N

Glucose (g/l) Glucose 10 (g/l) acid Citric 1

Condition O

0 0 0 1 2 3 4 5 6 Fermentation time (days) Figure 22- Bioreactor fermentation with I. orientalis in conditions N and O. Citric acid production in ondition N (orange circles) and condition O (blue triangles).

Citric acid concentration also reveals differences between the bioreactors (Figure 22). In the condition O, the citric acid concentration is kept below 1.2 g/l during all the fermentation time, while in the condition N, the concentration is between 2 and 3 g/l during all the process. As referred before, this result is in accordance with literature. Looking to the metabolic point of view, citric acid is a precursor of ITAC production. When the citric acid levels decrease in the extracellular medium, ITAC should increase if citric acid would only be converted into ITAC. However, this is not likely to occur because of the steps needed to convert citric acid into ITAC. At the day three of fermentation in the condition O, the citric acid concentration is reaching zero, but after the glucose feed on this third day, the concentration increases again. With engineered microorganisms, the best result for ITAC production is with E. coli (Harder et al. 2016) in a fed- batch system with 27 g/l of initial glucose concentration and addition of glucose periodically when glucose concentration was below 10 g/l. A productivity of 0.38 g. l-1. h-1 and carbon yield of 0.49 g/g was obtained by Harder et al. In the present work, 0.022 g. l-1. h-1 and 0.027 g/g of carbon yield was obtained. Different possibilities can explain so big differences, as the strain optimization for ITAC production and the period of cell starvation, since in this work, the glucose levels drop continuously until zero and only after some period of starvation, new carbon source is added to the medium.

4 Condition O

pH Condition N 2

0 0 1 2 3 4 5 6

Fermentation time (days)

Figure 23 – pH levels during the fermentation time with I. orientalis in bioreactor in conditions N (orange circles) and O (blue triangles)

The variation in pH for both conditions shows that the decrease of pH of the fermentation in condition N occurs mainly in the first 24 hours of fermentation, and that the pH control (condition O), does not let the pH drop under 4, and so, this can explain the differences obtained between the two conditions in the first day of fermentation. The constant pH over the fermentation time after day 1 for both conditions associated with the differences in ITAC production, suggest that pHd control at 4 is fundamental for the maintenance of the regular activity of the cells and so, responsible for higher ITAC production.

4.4.2 Bioreactors with cheese whey After test the conditions in shake flask, tests with bioreactors were done to check if the addition of the alternative sources could have a good behavior in ITAC production. Using cheese whey as a possibility to be a part of the medium for ITAC production, a bioreactor with 50% v/v of synthetic medium and cheese whey was tested. During these fermentation, the pH of the broth is controlled to 4 (condition P) and under the same conditions, a daily feed of glucose (20 g/l) was done (condition Q), as like the best condition obtained for synthetic medium. The results are displayed in Figures 24 and 25.

30 1.5

Citric acid

20 1

ITAC

glucose (g/l) 10 0.5

ITAC and citric acid(g/l)

0 0 0 1 2 3 4 5

Fermentation time (days)

Figure 24- Bioreactor fermentation with I. orientalis in condition P. Glucose consumption (grey squares), ITAC (blue triangles) and citric acid (orange circles) production during the fermentation with 50 % v/v synthetic medium and cheese whey

ITAC 2.5

) g/l 2 Citric acid 1.5

10 Glucose (g/l) Glucose

1 ITAC and citric acid ( acid citric and ITAC 0.5

0 0 0 1 2 3 4 5 Fermentation time (days)

Figure 25 – Bioreactor fermentation with I. orientalis in condition Q. Glucose consumption (grey squares), ITAC (blue triangles) and citric acid (Orange circles) production during the fermentation with 50 % v/v synthetic medium and cheese whey. For glucose consumption, the glucose added daily was not measured and it is considered the theoretical value of glucose upon addition. The values for time zero and 24h after every glucose addition were measured.

When no fed-batch was used (condition P), ITAC production achieved a best titre of 0.79 g/l and the citric acid concentration is above that value during all the fermentation time (Figure 24). The result can be comparable with the condition M, when with synthetic medium only and pH control, 0.63 g/l were obtained. In this condition P, 0.79 g/l were achieved in the same conditions but with 50% v/v cheese whey. No pH decreases under 4 was observed, so no base was added to the fermentation broth.

In condition Q, the higher titre of ITAC obtained was in the day five (last) of the fermentation (Figure 25). The result is very similar to the result obtained with synthetic medium for ITAC production, while the behavior for citric acid is slightly different. An important factor is that the pH of the fermentation broth never went down than 4, which means that no base was used to control the pH, but the cheese whey itself work as a buffer, never letting the pH decrease under 4.

The final concentration of ITAC in cheese whey bioreactor is approximately three-fold lower than in the shake-flask (2.66 g/l in bioreactor and 6.85 g/l in shake flask after five days of fermentation). In the bioreactor the conditions of fermentation are different from the shake flask, and since the carbon source is fast consumed by this strain in bioreactor (around 6h), the lack of sugar source for so many hours are maybe influencing ITAC production in a negative way. However, the use of cheese whey in bioreactor is possible and the results have potential to become more positive in further works. Table 7 summarizes all the conditions used in this work with I.orientalis.

Table 7-Summary of all the conditions and operations tested and ITAC titre, productivity, and carbon source yield.

Conditions Operation ITAC titre (g/l) Productivity Yield (g (g. l-1.h-1) ITAC/g glucose) Condition A Shake-flask batch 0.67 0.005 0.033 Condition B Shake-flask batch 0.64 0.005 0.016 Condition C Shake-flask fed-batch 1.13 0.009 0.028 Condition D Shake-flask fed-batch 0.98 0.008 0.025 Condition E Shake-flask fed-batch 2.1 (5 days) / 2.5 0.017/0.013 0.021/0.016 (8 days) Condition F Shake-flask fed-batch 1.6 0.013 0.016 Condition G Shake-flask batch 1.99 0.016 0.095 Condition H Shake-flask batch 0.76 0.006 0.038 Condition I Shake-flask batch 0.6 0.005 0.03 Condition K Shake-flask fed-batch 6.84 (5 days) / 0.057/0.051 0.068/0.061 9.83 (8 days) Condition L Batch Bioreactor 0.24 0.002 0.012 Condition M Batch Bioreactor with 0.63 0.005 0.031 pH control Condition N Fed-Batch bioreactor 0.33 (5 0.0027/0.0023 0.0033/0.0034 days)/0.34 (6 days) Condition O Fed-Batch bioreactor 2.73 (5 0.0227/0.019 0.027/0.027 with pH control days)/2.79 (6 days) Hydrolyzed Shake-flask batch 1.11 0.009 0.040 cheese whey Condition P Batch Bioreactor 0.79 0.007 0.04 Condition Q Fed-Batch bioreactor 2.67 0.022 0.026

4.5 Downstream strategy to recover ITAC production process Polybenzimidazole (PBI) is being used in iBB as an adsorber. This polymer is able to exchange anions or interact with neutral species. Considering the pKa of ITAC as main product and citric acid as by-product, the effect of pH on separation by different absorbers was tested. The pKa of ITAC and citric acid are in Table 8.

Table 8- pKa values for ITAC and citric acid.

Organic acid pKa Citric acid 3.13; 4.76; 6.40 ITAC 3.84; 5.55

When the fermentation broth is at pH 3.13, theoretically, 50% of citric acid is in the protonated state and the other half in the first deprotonated state, while ITAC is mostly in the protonated state. Regarding the results in bioreactor, with pH control at pH 4 is the most advantage condition, and thinking about a downstream operation in this bioreactor, separations at pH 4 are interesting. In this test, commercial ITAC and citric acid were used to test the capability of PBI to separate one of the acids in the resin and let the other in the solution or both at the same time.

PBI and commercial Amberlite IRA458, an acrylic gel type strongly basic anion exchange resin, were used (Table 9 and 10). Solutions of 20 g/l of ITAC and 40 g/l of citric acid were used and pH of 1.8, 3.5,4.7 and 7 were used for the tests.

Table 9 – Samples of ITAC and citric acid were used in PBI for these acids recover.

Sample + PBI % citric acid in % of itaconic % citric acid % itaconic acid in solution acid in solution in resin resin pH 1.8 (original pH) 96.3 78.2 3.8 21.8 pH 3.5 99.5 78.7 0.5 21.3 pH 4.7 82.1 75.2 17.9 24.8 pH 7 76.0 61.3 24.0 38.7

Table 10 - Samples of ITAC and citric acid were used in IRA458 for these acids recover.

Sample + IRA-458 % citric acid in % of itaconic % citric acid % itaconic acid in solution acid in solution in resin resin pH 1.8 (original pH) 90.4 92.7 9.6 8.3 pH 3.5 80.9 86.2 19.1 13.8 pH 4.7 82.1 76.5 17.9 23.5 pH 7 37.0 28 63.0 72

Both PBI and Amberlite IRA458 seem to follow the theoretical pathway of adsorption, since when the pH of the solution is increased, i.e., when the organic acids are in their deprotonated state, more citric and ITAC acids are attached to the adsorption polymer and resin. However, the results regarding the separation shows that is not selective enough to promote separation between the two organic acids. However, the adsorbed can be used to recover both organic acids from the aqueous solution/fermentation broth.

5. General conclusions This work provides new information about the behavior of I. orientalis in bioreactor and using alternative substracts that can be used as carbon source for ITAC production. To evaluate the capacity of the strain to react to different sugar source concentrations and feed strategies, tests were performed regarding the initial carbon source concentration and the addition of the same during the fermentation. The non-conventional yeast I. orientalis seems to react positively to pulse feeding strategy: daily carbon source feeds with continuous increase of ITAC concentration in the fermentation broth.

The decrease of production costs of ITAC is of urgent concerning concerning the possible markets where ITAC can be placed in the next years. The biotransformation of waste into valuable fine chemicals was made in this work with cheese whey as substrate to produce ITAC with I. orientalis. The study excluded cheese whey as a direct carbon source for I. orientalis and S. cerevisiae since these strains are not able to metabolize lactose. However, when lactose is hydrolyzed into its monomers, glucose and galactose, I. orientalis is able to grow and ITAC to be produced, with approximately 1.12 g/l of ITAC produced from 27 g/l of glucose.

Using cheese whey as a complementary source for a rich media (without hydrolysis) for I. orientalis, the best result in shake-flasks was achieved, with 9.83 g/l of ITAC produced in a fed- batch system from a total of 160 g/l of glucose, in comparison with the synthetic medium, where only 2.49 g/l of ITAC were achieved in the same conditions. This condition is then interesting to be tested in bioreactor regarding ITAC production.

The presence of a peak in HPLC analysis for day zero of fermentation for the retention time of ITAC and citric acid, among others when cheese whey was used needed to be clarified regarding the authenticity of ITAC production with cheese whey in the medium. A simple test with I.orientalis with and without the inserted plasmid that has the gene that codifies for the enzyme CAD, responsible for cis-aconitate conversion to ITAC reveals that what is being produced is ITAC and no other metabolite that I.orientalis is capable of synthesize.

The use of bioreactors for ITAC production with I.orientalis is not of previous knowledge. The approaches used in this work showed that pH control is fundamental to produce more ITAC. pH controlled at 4 showed 3-fold the concentration of the bioreactor with no control. Fed-batch strategies were also tested in both controlled and non-controlled pH conditions. A final concentration of 2.7 g/l of ITAC was obtained in the controlled bioreactor, against 0.34 g/l in the non-controlled bioreactor. Cheese whey was also used as a rich-medium for bioreactor after the good results obtained in shake-flask, in a fed-batch system. The results are very similar for ITAC production in cheese whey and in the synthetic medium bioreactor with pH control at 4. The pH of the bioreactor with cheese whey never dropped under 4.5. This can mean that use cheese whey or use base in the bioreactor seems to be the same and that in shake-flask, cheese whey acted as a pH control and not as rich-medium. This is a bottleneck on using cheese whey as a

47 rich medium but still, the use of cheese whey as the fermentation medium itself after the lactose break seems to be efficient.

The separation of ITAC from the fermentation broth to obtain purified and concentrated ITAC is from the highest interest. PBI was tested as an adsorber with the aim of separate ITAC from citric acid or both from the aqueous solution/fermentation broth. The results regarding the separation shows that is not selective enough to promote separation between the two organic acids. However, the adsorbed can be used to recover both organic acids from the aqueous solution/fermentation broth.

6. Future perspectives New studies can be performed based on the work described in this thesis. The use of I. orientalis with alternative substrates can be done since this microorganism showed higher tolerance to different media with different toxic compounds profile. In addition, the study of the other media components would have interest for the substitution or deletion of synthetic medium components used in this work. Metabolic engineering strategies for lactose breakdown and/or galactose utilization would be an advantage regarding cheese whey directly use as a medium for I. orientalis fermentation. The study of the intracellular ITAC by per example disrupt the cells and calculate the total amount before and after the disruption would allow to know if some produced ITAC is still in the interior of the cells and some work can be created to minimize this amount. The understanding of the modification of citric acid levels in the extracellular medium when glucose is added is another important work that can be followed. The behavior of citric acid in the fermentations in fed-batch has no explanation with the methodologies used in this work.

Regarding the ITAC downstream purification, the use of PBI as a polymer for ITAC and citric acid separation from aqueous solution/fermentation broth needs further development regarding the use of this PBI as an alternative for a downstream process. The use of different solution/polymer ratios should be addressed in further work. The elution of the ITAC and citric acid that were adsorbed need to be done by changing the pH of the solution to change the protonated states of both acids and make them elute. The use of solvents with affinity for only one of the acids can be the way to separate ITAC from citric acid in a two-step protocol with polymer adsorption and selective elution.

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Appendix 1

Annex 1 - Fermentation with S. cerevisiae in 1.5 L vessel bioreactor

0.030

0.025

0.020

0.015

ITAC ITAC (g/L) 0.010

0.005

0.000 0 1 2 3 4 5 6 7 8 9 10 Fermentation time (days)

A1.1 – ITAC production

Galactose Galactose (g/L) 10

0 0 1 2 3 4 5 6 7 8 9 10

Fermentation time (days)

A1.2 - Galactose consumption

Acetic Acetic Acid(g/L) 10

0 0 1 2 3 4 5 6 7 8 9 10 Fermentation time (days)

A1.3 – Acetic acid

Appendix 2 CSM, vitamins and trace elements. Retrieved from Campos, AVS Thesis (2015)