Novel Insight for the Production of Polyhydroxyalkanoates from Renewable Resources

Rodrigo dos Santos Raposo

Dissertação para a obtenção do Grau de Mestre em Engenharia Biológica

Júri

Presidente: Profª. Maria Raquel Murias dos Santos Aires Barros Orientadores: Profª. Maria Manuela Regalo da Fonseca Dr. Dipl.-Ing. Martin Köller Vogal: Profª Catarina Dias de Almeida

Dezembro 2009

Abstract

The current work is a cluster of experiments where the main goal is to enhance the PHAs productivity by using different approaches. In the first experiment and using Bhurkholderia sacchari DSM 17165, the maximum production of PHB achieved was around 45% of the total cell dry mass (7.6 g/l) when grown with sucrose as carbon source. In the second experiment and using both strains Pseudomonas putida KT 2442 and Pseudomonas oleovorans ATCC 29347 the best growth results were obtained by using Oleic acid with both strains in comparison to saponified olive oil or dodecanoate. Although for a bigger scale experiment, the precursor used should be saponificated olive oil considering that the slight differences obtained with oleic acid do not justify the increased cost of this acid when compared to saponificated olive oil. In the third experiment and following the conclusions of the previous experiment the same both strains were grown, at a bioreactor scale, on glucose and, after a reasonable cell density was obtained, the cultures where fed with oleic acid. Some obstacles appeared with the execution of the experiment with the strain P.oleovorans ATCC29347, making it clear that the pH plays a fundamental role in the development of the culture, more precisely on the growing kinetics of the bacteria. By using the strain P. putida KT2442, a maximum cell dry mass of 27 g/l and a final cell dry mass of 18 g/l was achieved, the microscope analysis proved the existence of vast amounts of PHA inclusions. The final experiment is the direct result of the necessity to lower the cost as well as to enlarge the amount of current carbon sources available for the production of PHAs, taking into consideration that the carbon source costs at an industrial scale usually represent above 50% of the global production costs. In this experiment a series of three waste materials from the olive oil and tomato pulp industry, were submitted to a series of acid hydrolysis, in order to convert them into fermentable sugars. The best result was obtained with a type of olive oil waste (Marc 2), by producing a total of 5.19 g/l of fermentable sugars corresponding to 13% of w/w conversion from the raw material.

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Resumo

O presente trabalho consiste num conjunto de experiências cujo principal objectivo é o aumento da produtividade de PHAs através da utilização de diferentes abordagens. Na primeira experiência e usando a estirpe Burkholderia sacchari DSM17165 a produção máxima de PHB atingida foi de cerca de 45% da biomassa seca total ( 7.6 g/l ) quando sacarose foi utilizada como fonte de carbono. Na segunda experiência e usando as estirpes Pseudomonas putida KT2442 e Pseudomonas oleovorans ATCC29347, os melhores resultados, relativamente ao crescimento de biomassa, foram obtidos através da utilização de ácido oleico em comparação com azeite saponificado ou dodecanoato. Aquando da execução de uma experiência em maior escala, o precursor a ser utilizado deveria ser azeite saponificado, tendo em conta que as ligeiras diferenças obtidas com o ácido oleico não justificam o maior custo deste ácido comparativamente ao azeite saponificado. Na terceira experiência e em consequência das conclusões obtidas na experiência anterior, as mesmas estirpes foram cultivadas, numa escala de bio-reactor, usando glucose. Após se ter atingido uma densidade celular razoável as culturas foram alimentadas com ácido oleico. Apesar de alguns obstáculos terem surgindo aquando da execução da experiência com a estirpe P. oleovorans ATCC29347, tornou-se óbvio que o pH desempenha um papel fundamental no desenvolvimento da cultura, mais precisamente na cinética de crescimento das bactérias em causa. Atingiu-se uma concentração máxima de 27 g/l e uma concentração final de biomassa de 18 g/l aquando da utilização da estirpe P. putida KT 2442 , a observação ao microscópio revelou a existência de inclusões de PHA. A experiência final é o resultado directo da necessidade em diminuir o custo assim como alargar a quantidade actual de fontes de carbono disponíveis para a produção de PHAs. Tendo em conta que o custo dessa fonte de carbono, a escala industrial normalmente representa mais de 50% do custo total de produção. Nesta experiência uma série de três desperdícios da indústria do azeite e o tomate, foram submetidos a uma série de hidrólises ácidas com o objectivo de os converter em açúcares fermentáveis. Os melhores resultados foram obtidos com um tipo de desperdício de azeite (Marc 2), tendo sido atingida uma produção total de 5.19% em açúcares fermentáveis correspondendo a uma conversão de 13% (massa/massa) da matéria prima.

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Acknowledgement

First of all I would like to thank to all the students and assistants from the TU Graz for their friendship and support during my internship, making my Erasmus experience as pleasant and rewarding as it could be. I am especially grateful to Martin Koller, Professor Braunegg and Miguel Dias for their guidance, teaching and helping throughout the project. I would also like to thank Anna Salerno for her assistance and help during the last part of my work. I also had a big support from my home University, so I would like to thank the opportunity it gave me to do the final internship in a foreign Institution and particularly to Prof. Manuela Fonseca, who always offered her help during the project and supported me greatly with the manuscript. Finally, I want to express my deep gratitude to my family, especially my parents and sisters, my close friends and university colleagues for all the assistance given during my universitary period.

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Publications

Koller M., Miranda de Sousa Dias M., Reiterer A., Salerno A.,dos Santos Raposo R.; Braunegg G., Biopolyester aus Nawaros, 52, 2009, 5

Koller M., Salerno A. Ingolic E., Miranda de Sousa Dias, Reiterer A M., Raposo R.; Braunegg G., Contexts between the bio-mediated production of polymers, chemicals, and fuels from waste streams, Biologi italiana, 8, 2009, 8 - 11

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Table of contents

Part 1: Fermentation Enhancements for the Production of Short and Medium Chain Length Polyhydroxyalkanoates...... 10

1. Introduction...... 10

1.2. Biodegradable plastics – Past, Present and Future...... 11 1.3.Properties of PHAs...... 12 1.4.Use and applications of PHAs...... 13 1.5.Synthases...... 15 1.6.Strains...... 18 . 2. Growth evolution of the strain Burkholderia sacchari DSM 17165 under different carbon source combinations...... 23

2.1 Materials and Methods...... 23 2.2.Results...... 25 2.3.Conclusions ...... 30

3. Determination of the growth kinetics of both strains Pseudomonas oleovorans ATCC 29347 and Pseudomonas putida KT under different fatty acids...... 32

3.1.Materials and methods...... 32 3.2.Results ...... 34 3.3 Conclusions...... 39

4. Growth behaviour at a Bioreactor scale of the strains Pseudomonas oleovorans ATCC 29347 and Pseudomonas putida KT 2442 using the precursors oleate and dodecanoate...... 40

4.1.Materials and Methods...... 40 4.2.Results...... 43 4.3.Conclusions...... 48

Part 2 : Acid Hydrolysis for the Production of Fermentable Sugars from Waste Raw Materials...... 49

5.1Introduction...... 49 5.1.1.Hemicellulose removal – Xylan removal...... 50 5.1.2.Xylose degradation...... 52 5.1.3.Cellulose breakdown and glucose degradation...... 53 5.1.4.Cellulose breakdown vs hemicellulose breakdown...... 55 5.2.Acid hydrolysis for the production of fermentable sugars from complex raw materials...... 57 5.3.Conclusions ...... 68

6. Final considerations...... 69 6. Bibliography...... 70

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List of figures

Figure 1. The best known and probably most widely distributed (P-HB) metabolic pathway, which also occurs in C. necator, Z. ramigera, and A. Beijermckii ( Seebach et al. 1993)

Figure 2. Shampoo bottles made of BIOPOL, after 0, 3, and 9 months in compost. ( Seebach et al. 1993)

Figure 3. Examples of Constituents that can be found in PHAs (Steinbüchel, 2001)

Figure 4. The three classes of PHA synthases identified so far, subunits that compose each enzyme, natural occurrence in microorganisms and substrates these enzymes act on (Steinbüchel, 2001)

Figure 5. Phylogenetic tree of the species Bhurkholderia sacchari (Steinbüchel et al , 2001)

Figure 6. Microscope image of the strain P. putida KT 2442 with polymer inclusions when grown on dodecanoate

Figure 7. Composition of PHA synthesized by P. putida KT 2442 during growth on different carbon sources (Wltholt et al 1991)

Figure 8. Influence of cultivation temperature on PHA composition in P. putida (Witholt et al, 1991)

Figure 9. Graphic representation of the xylan removal curve as the result of the sum of two linear curves (Zerbe et al , 1985)

Figure 10. Schematic representation of the xylose degradation pathway (Zerbe et al 1985)

Figure 11. Typical chromatogram obtained from the HPLC analysis of a xylan hydrolysis sample (Zerbe et al, 1985)

Figure 12. Values for the constants P1 and P2 dependant on the acid concentration, values for the activation energies ∆H1 and ∆H2( Yan et a,l 2008)

Figure 13.Cellulose hydrolysis model (Zerbe et al ,1985)

Figure 14. Graphic representation of the hemicellulose breakdown constant k, dependant on acid concentration, at 120ºC(Zerbe et al ,1985)

Figure 15. Graphic representation of the cellulose breakdown constant k c, dependant on acid concentration, at 180ºC(Zerbe et al ,1985)

7 List of tables

Table 1. Compounds, respective concentration and mass weighed to prepare the pre culture media

Table 2. Compounds, respective concentration and mass weighed to prepare the fermentation media

Table 3. Media identification according to the sugars used

Table 4. Average calculations of specific growth rate, final cell dry mass concentration, final PHB concentration and final PHB percentage of the 7 different media

Table 5. compounds, respective concentration and mass weighed to prepare the pre culture media

Table 6. compounds, respective concentration and mass weighed to prepare the fermentation media

Table 7 and 8. Media description and respective fatty acid contents for each individual fermentation

Table 9. Compounds and respective concentration used to prepare the pre culture and inoculum media

Table 10. Compounds and respective concentration used to prepare Reactors A and B media

Table 11. Glucose, ammonia hydroxyde and oleate additions performed on reactor 3

Table 12. Glucose, ammonia hydroxyde and oleate additions performed on reactor 4

Table 13. Time, Glucose,Xylose and Fructose concentrations and Fermentable sugar yield for each sample taken in the second acid hydrolysis

Table 14. Time, Glucose,Xylose and Fructose concentrations and fermentable sugar yield for each sample taken in the acid hydrolysis following the Mosier method

Table 15. Time, Glucose,Xylose and Fructose concentrations and fermentable sugar yield for each sample taken in the Marc 1 acid hydrolysis following the Saeman method

Table 16. Time, Glucose,Xylose and Fructose concentrations and fermentable sugar yield for each sample taken in the Marc 2 acid hydrolysis following the Saeman method

Table 17. Time, Glucose,Xylose and Fructose concentrations and fermentable sugar yield for each sample taken in the Tommato waste acid hydrolysis following the Saeman method

List of graphics

Graphic 1. Set of graphics that show the evolution of sugars and ammonia concentration for the 14 individual fermentations

Graphic 2. Set of graphics that show the time curves of cell dry mass, residual biomass and PHA concentration for the 14 individual fermentations

Graphic 3. Set of graphics that show glucose and ammonia concentrations for the fermentations performed by the strain P. oleovorans ATCC 29347

8 Graphic 4. Set of graphics that show glucose and ammonia concentrations for the fermentations performed by the strain P. putida KT 2442

Graphic 5. Optical density measured in each fermentation for the strain P.oleovorans ATCC 29347

Graphic 6. Cell dry mass and measured in each fermentation for the strain P.oleovorans ATCC 29347

Graphic 7. Optical density measured in each fermentation for the strain P. putida KT 2442

Graphic 8. Cell dry mass and measured in each fermentation for the strain P. putida KT2442

Graphic 9. Cell dry mass concentration and optical density values observed on reactor 3

Graphic 10. Glucose concentration, glucose and oleate additions over time for reactor 3

Graphic 11. Ammonia concentration over time on the reactor 3

Graphic 12. Cell dry mass concentration and optical density values observed on reactor 4

Graphic 13. Glucose concentration, glucose and oleate additions over time for reactor 4

Graphic 14. Ammonia concentration over time on the reactor 4

Graphic 15. HPLC chromatogram of the first acid hydrolysis performed, 5 g of Marc 1, acid concentration of 6M, temperature of 90ºC

Graphic 16. HPLC chromatogram of the second acid hydrolysis performed, 20 g of Marc 1, acid concentration of 6M, temperature of 105ºC, rose curve (Sample 1), blue curve (sample 3), black curve (Sample 5), brown curve (sample

Graphic 17. HPLC chromatogram of hydrolysis following the Mosier method , 15 g of Marc 1, acid concentration of 50 mM, temperature of 160ºC, black curve (Sample 2), rose curve (sample 3),blue curve (Sample 4)

Graphic 18. HPLC chromatogram of cellulose hydrolysis following the Saeman method , 10 g of Cellulose, acid concentration of 100 mM, temperature of 180ºC, black curve (Sample 2), rose curve (sample 3),blue curve (Sample 4)

Graphic 19. HPLC chromatogram of Marc 1 acid hydrolysis following the Saeman method , 20 g of Marc 1, acid concentration of 100 mM, temperature of 180ºC, black curve (Sample 2), rose curve (sample 4),blue curve (Sample 5)

Graphic 20. HPLC chromatogram of Marc 2 acid hydrolysis following the Saeman method , 20g of Marc 2, acid concentration of 100 mM, temperature of 180ºC, black curve (Sample 2), rose curve (sample 4),blue curve (Sample 5)

Graphic 21. HPLC chromatogram of tomato waste acid hydrolysis following the Saeman method , 20 g of tomato waste, acid concentration of 100 mM, temperature of 180ºC, black curve (Sample 3), rose curve (sample 4),blue curve (Sample 6), blue curve (Sample 7)

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Part 1: Fermentation Enhancements for the Production of Short and Medium Chain Length Polyhydroxyalkanoates

1. Introduction

Polyhydroxyalkanoates (PHAs) are synthesised by a variety of microorganisms under conditions of nutrient limitation when in the presence of an excess of carbon or energy source. Due to their low solubility in water and high molecular weight, PHAs are ideal storage compounds since they do not cause an increase in the intracellular osmotic pressure. Under this role these compounds are much more common within microorganisms than glycogen, polyphosphates or fats.

The most common of the PHAs, polyhydroxybutyrate P(3-HB), was first described by Lemoigne in 1925 who later isolated and identified the material from BaciIlus megaterium . Since then P(3-HB) as well as a wide variety of other PHAs have been discovered in a large number of different microorganisms. Although the presence of PHAs has been described in superior organisms such as animals and plants in low molecular weight forms where it presumably forms part of an ion channel and more recently in the human blood plasm where it is bound mainly to the so-called “low density lipoproteins” and to albumin, these putative functions of the PHAs are not of interest to the present work and will not be further discussed.

Quantitatively, the most important role of PHAs is to store carbon-containing material and reductase equivalents in the cells of prokaryotic microorganisms, for P(3-HB) commonly known as simply PHB, values up to 90% of the cell dry mass have already been reached in some specific strains of some species namely some strains of Cupriavidus necator. The biochemistry of the synthesis and degradation of high molecular weight PHAs has been subjected to intensive studies which have demonstrated which other hydroxy acids can be incorporated into the polymer, in natural or synthetic growth media. The genes responsible for the enzymatic machinery involved in the polymer biosynthesis have been identified and incorporated into other species.

PHAs occur in the cytoplasm of the cell in the form of inclusion bodies, so-called granules. Typically, these have a diameter of 100-800 nm, and have been shown to be surrounded by a kind of micelle (“monolayer”), but not by a double layered structure (typical membrane, lipoprotein bilayer). Depending on the bacteria, the PHA synthase as well as the depolymerase system may also be bound to this envelope. Purified granules of the species B. megaterium consist of 97.7% of P(3-HB) being the remaining composed of proteins and lipids.

Intracellular Poly(3-hydrobutyrate) metabolism is a cyclic process, involving seven enzymes as represented on the following scheme

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Figure 1 . The best known and probably most widely distributed (P-HB) metabolic pathway, which also occurs in C. necator, Z. ramigera, and A. beijermckii ( Seebach et al. 1993)

1.2. Biodegradable plastics – Past, Present and Future

Plastics make up about 7% by weight and over 30% by volume of the total garbage produced today in the USA and Japan. This can be explained in part by the fact that plastics are more and more replacing glass, paper, or metal as packaging materials. In principle, there are three possibilities for the disposal of plastics: incineration, recycling, and degradation in the environment. Unfortunately all of them pose serious practical problems. Incineration releases highly polluent compounds in the atmosphere. Recycling is usually much more expensive than simply producing new plastic. Environmental degradation is not yet possible for many of the petrochemical plastics, at least in a reasonable period of time (Seebach et al, 1993).

International policies driven by an increasing popular environmental conscience, mostly in USA , Europe and Japan, are turning the table in favour to biodegradable plastics. Most of the European Ambientalist / Ecological political parties are gaining strength, and applying pressure in order to ratificate laws that force the industry and society to bet on these biodegradable polymers.

11 The bioplastic market moved around 61 million pounds in the year 2000, around 114 million pounds in the year 2000 and it is expected to move more than 206 million by the year 2010.

The USA is responsible for the production of 30% of all the biopolymers and Japan responsible for 10%. Although the estimate of 20% per year growth rate for these plastics market done during the 1990’s was not accomplished due to a low capacity in new companies to thrive in this market, it is most likely that the political and economical conjecture will lead the biopolymer industry to a bright future, and therefore responsible research in this field should be genuinely valuable.

1.3.Properties of PHAs

All PHAs possess some properties which recommend them for some applications and make them interesting to industry such as:

• They are thermoplastics and/or elastomeric compounds which can be processed with apparatus already used by the plastic manufacturing industry to mimic some of the most important petrochemical polymers. • Insoluble in water • Exhibit a rather high degree of polymerization ranging from 10 5 to almost 10 7 Da • They are enantiomerically pure chemicals consisting in general, only of the R- stereoisomer. • Non-toxic • Biocompatible • At least poly(3HB) and poly(3HB-co-3HV) possess piezoelectric properties

• Several PHAs can be obtained from CO 2 or renewable resources • All PHAs are biodegradable

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1.4.Use and applications of PHAs

Commercial production of PHA is so far only viable by fermentative biotechnological processes. Several PHAs, such as, in particular, the homopolyester poly(3HB), the copolyester poly(3HB-co-3HV) and PHAs consisting of 3-hydroxyoctanoate, 3-hydroxydecanoate and a few other medium-chain-length 3-hydroxyalkanoates, poly(3HA MCL ), have been manufactured into various materials, and applications in various areas have been revealed. Whereas the use of poly(3HB) and poly(3HB-co-3HV) as biodegradable bioplastics was established some time ago, other applications such as the manufacturing of latex paints, and specifically medical applications including retard materials and use as scaffolding material for tissue engineering due to their biocompatibility, are currently under development (Steinbüchel, 2001).

Isolated and purified PHAs can be used as such or in combination with other materials such as starch, cellulose fibers, glass fibers or synthetic plastics to obtain compounded materials.

The purified material can be processed through transterification, melted in the presence of synthetic polyesters, by modification of the side chains or by crosslinking through energized irradiation and chemical reagents. Another usage of the PHAs can be done as a source to obtain enatiomeric pure hydroalkanoic acids upon chemical or enzymatic cleavage for the synthesis of various chemicals. There have also been applications for bacterial cell mass containing a high fraction of PHAs as binders for other fibrous materials (Steinbüchel, 2001)..

Other applications for such polyesters can be found in agriculture (e.g. as cold frame sheeting, seed capsules, or formulations for the controlled release of pesticides or herbicides”). A process for elimination of nitrates from drinking water using PHB has also been developed. (Steinbüchel, 2001).

Recently attempts have been made to extend the field of application of P(3-HB) and P(3- HB/3-HV) by preparing degradable block copolymers with polystyrene and polyethers, or “blends” with polyethylene, polystyrene, polyvinyl chloride, or polyethylene oxide act as a gas barrier in a similar way to polyvinyl chloride or polyethylene terephthalate. Because of these properties, P(3- HB) could well play an important role as a packaging material. Nowadays this is the only widespread application of this material, mostly used to produce shampoo bottles that degrade themselves within a compost in a short span of time (Steinbüchel, 2001).

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Figure 2. Shampoo bottles made of BIOPOL, after 0, 3, and 9 months in compost ( Seebach et al. 1993).

Judging by the increasing number of publications, the area is in the midst of great developments. How well PHAs can assert themselves against other biopolymers remains to be seen .

14 1.5.Synthases

Approximately 150 different hydroxyalkanoic acids are presently known as constituents of PHA storage polyesters. Some of these constituents can be seen in figure 3. That demonstrates the variability of the structures and sizes of polyhydroxyalkanoic acids detected in PHAs .

Figure 3. Examples of Constituents that can be found in PHAs (Steinbüchel, 2001)

Hydroxyalkanoates are synthesized by diverting intermediates of the carbon metabolism to hydroxyacyl-CoA thioesters. The latter are then polymerized by PHA synthases which are bound to the surface of PHA granules together with other proteins. However, most of these PHAs are only obtained if precursor substrates, which are structurally more or less related to the constituents to be incorporated into PHAs, are provided as a carbon source for the bacteria, because central intermediates can not be diverted to the respective hydroxyacyl-CoA thioesters. Only a few PHAs

15 can therefore be obtained from simple carbon sources which are available in large amounts from agriculture, such as carbohydrates and fatty acids, or from the CO2 of the atmosphere.

Figure 4. The three classes of PHA synthases identified so far, subunits that compose each enzyme, natural ocurrence in microorganisms and substrates these enzymes act on (Steinbüchel, 2001)

PHA synthases can be classified into three different classes according to their structure and substrate range. Class I comprises PHA synthases consisting of only one type of subunit with molecular weights ranging from approximately 60 to 65 kDa in most cases. Another characteristic of class-I PHA synthases is that predominantly hydroxyalkanoic acid with three, four or five carbon atoms are incorporated into PHAs. This class is represented by the PHA synthase of C. necator and occurs in all other poly(3HB) accumulating bacteria (Steinbüchel, 2001).

Class II comprises PHA synthases also consisting of only one type of subunit and with a similar molecular weight range; however, the substrate specificities of PHA synthases belonging to this class are different. These PHA synthases incorporate medium-chain-length 3-hydroxyalkanoic acids into PHAs. The chain length characteristically comprises 6 to 14 carbons with a high freedom for the structure of the R-pendant group. This class is represented by the enzyme of Pseudomonas oleovorans and seems to occur exclusively in pseudomonads sensu strictu (Steinbüchel, 2001).

Finally the class III enzymes comprises PHA synthases consisting of two different types of subunits each with a molecular weight of approximately 40 kDa. One of the subunits exhibits

16 significant homologies with the class I and class II synthases. PHA synthases of this class occur in all cyanobacteria and all sulphur purple bacteria but not in the non-sulphur bacteria investigated so far. The class-III PHA synthases of Chromatium vinosum and Thiocapsa pfennigii have been studied in greatest detail. One reason for this was the unusual structure of this PHA synthase. The other reason was the unusual substrate range. For the PHA synthase of T. pfennigii , it was demonstrated that the enzyme was able to synthesize copolyesters of 3HB and various medium- chain-length hydroxyalkanoic acids, even those with the hydroxyl group not at the 3’ position (Steinbüchel 2001).

PHA synthases that accept as substrate monomers with a chain length higher than 14 carbons have never been observed, thus PHAs containing this type of monomer were never obtained as a result of biosynthesis. It is probably possible to produce them under laboratory conditions as a result of chemical synthesis, but at this point its commercial viability is surely inexistent.

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1.6.Strains

As previously shown, much work regarding the short chain length (scl) polymer producing strains was already performed by the scientific community and a wide variety of interesting results was already obtained.

The challenge facing this type of strains is not so much devoted to obtaining higher yields of polymer or higher cell densities, but to lower the overall production cost of, at least the PHB. There are many approaches to this problem, one of them has to do with the reduction of the substrate cost especially the carbon source which represents more than 50% of the total production cost of the PHB in large scale. As mentioned before only this polymer is produced at an industrial scale, and one of the facilities involved in this industry is located in Brazil, an industrial facility, coupled to a sugar cane mill, that uses part of the produced sugar to produce the polymer (Brazil PHB).

However the species used at this facility is Cupriavidus necator, deeply studied and capable of accumulating polymer up to 90% of the total cell dry mass. The problem is that this strain cannot feed directly on sucrose, the common sugar obtained directly from the sugar cane milling process. Therefore the process must have an initial step which consists in hydrolysing the sucrose into glucose and fructose, sugars that C. necator is capable of assimilating.

In the same soils where the cane is planted, a species latter indentified as Burkholderia sacchari was discovered capable of feeding directly from sucrose (possibly this species secretes an enzyme that breaks down the sucrose).This species if properly studied, and if the results obtained are satisfactory enough could replace the C. necator on all the industrial processes where this strain is used to produce polymer, taking into consideration that sucrose has a much lower cost than both glucose or fructose.

With the proper study and experiments this species could also be used for the production of Poly(3HB-co-3HV), from sucrose and propionic acid. According to the literature the name of this strain Burkholderia sacchari [sac’cha.ri. N.L. adj. sacchari of sugar, referring to the location of isolation of the strain, soil of a sugar-cane (Saccharum officinarum)plantation]is due to the place where it was first discovered (Steinbüchel et al. 2001).

The phylogenetic tree of this strain can be seen on the figure 5, the information was obtained through the 16s rna analysis.

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Figure 5. Phylogenetic tree of the species Burkholderia sacchari (Steinbüchel et al . 2001)

Colonies of strain IPT101T on nutrient broth medium (Difco) are white and opaque due to the accumulation of poly(3HB) and poly(3HB-co-3HV). Cells are Gramnegative and are lysed by 3% KOH, motile due to the presence of several polar Flagella, rod-shaped (0.5- 0.8 µm in width, 1.5-3.0 µm in length) and grow well between 25 and 37 °C with an optimum growth temperature of 28-30 °C. No growth is detectable over a time period of 10 d at temperatures of 7 or 42 °C. Spores are not observed. Oxidase and catalase are produced. Nitrate is reduced to nitrite. Liquefaction of gelatin and hydrolysis of aesculin are not observed (Steinbüchel et al . 2001).

The following substrates are assimilated: acetate, acetylglucosamine, adipate, adonitol, dl- arabinose, d-arabitol, citrate, d-fructose, dl-fucose, galactose, gluconate, d-glucose, glycerol, inositol, 2-ketogluconate, lactate, d-lyxose, malate, mannitol, d-mannose, phenylacetate, propionate, pyruvate, draffinose, sorbitol, sucrose and d-xylose. The following carbon sources are not used: starch, amygdalin, larabitol, arbutin, caprate, cellobiose, dulcitol, erythritol,methyl α-d- glucoside, methyl α-d-mannoside, β-gentiobiose,glycogen, inulin, 5-ketogluconate, lactose,maltose, melezitose, melibiose, methyl β-xyloside,rhamnose, ribose, salicin, l-sorbose, d-tagatose, trehalose,d-turanose, xylitol and l-xylose. The following carbohydrates are oxidized: adonitol, dl- arabinose,d-arabitol, d-fructose, dl-fucose, galactose, gluconate,d-glucose, glycerol, inositol, 2- ketogluconate, d-lyxose,mannitol, d-mannose, d-raffinose, ribose, sorbitol,sucrose and d-xylose. The following carbohydrates are not oxidized: N-acetylglucosamine,starch, amygdalin, l-arabitol, arbutin, cellobiose, dulcitol, erythritol, b-gentiobiose, glycogen, inulin, 5-ketogluconate, lactose, maltose, melezitose, melibiose,methyl α-d-glucoside, methyl α-d-mannoside, methyl β-xyloside,

19 rhamnose, salicin, l-sorbose, d-tagatose, trehalose, d-turanose, xylitol and l-xylose. Strain IPT101T is susceptible to the antibiotics tetracycline (100 µg/ml), kanamycin (50µg/ml), chloramphenicol (100µg /ml) and ampicillin (15 µg /ml) (Steinbüchel et al , 2001).

The strain described in the literature is possibly the same as the one used during this work (B. sacchari DSM 17165) but belonging to a different collection, or a similar strain. In any of the cases the information collected should apply to the strain B. sacchari DSM 17165.

Fluorescest pseudomonads strains are unable to accumulate PHB, but they share the ability to produce medium chain length PHAs during unbalanced growth on medium and long chain alkanols and fatty acids.

Figure 6. Microscope image of the strain P. Putida KT2442 with polymer inclusions when grown on dodecanoate

The PHA biosynthetic pathway is not yet fully elucidated, but it has been proposed that 3- hydroxyacyl-CoA intermediates of the beta-oxidation pathway for the degradation of medium- and

20 long-chain alkanols and fatty acids are substrates for the PHA polymerizing system. (Witholt et al, 1991). During growth on medium chain length fatty acids, Pseudomonas putida accumulates PHA containing 3 hydroxy fatty acids which are, with respect to the carbon chain length whether directly derived from the substrate or shortened by one or more C2 units. (Witholt et al, 1991)

It was generally assumed that PHAs could only be formed in pseudomonads cells growing on medium or long chain fatty acids. It was found that P. putida also accumulates PHA during unbalanced growth on glucose and other unrelated substrates to alcohols or fatty acids (Whiltholt et al 1991). The same authors demonstrated that several pseudomonas strains, when grown on nonrelated substrates, accumulate PHAs which consist predominantly of 3-hydroxydecanoate monomer units. Minor constituents of these polymers were 3-hydroxyhexanoate, 3- hydroxyoctanoate and 3 hydroxydodecanoate. (Witholt et al, 1991)

The specific strain used from the Pseudomonas putida species was P. putida KT 2242. Some intensive studies with this strain were performed in the past and the composition of the PHA produced by this strain determined under varied cultivation conditions. The results vary from substrate used and even temperature

Figure 7. Composition of PHA synthesized by P. putida KT2442 during growth on different carbon sources (Witholt et al, 1991)

Figure 8. Influence of cultivation temperature on PHA composition in P. putida (Witholt et al, 1991)

21

The other medium chain length polymer producing strain used was the P. oleovorans ATCC 29347, a strain with the OCT plasmid, that makes it grow and produce polymer feeding preferably on octane, from the Pseudomonas oleovorans complex. This strain, when fed on octanoate, accumulated a blend of the homopolyester 3HB and a copolyester consisting of 3- hydroxyhexanoate and 3-hydroxyoctanoate (Rehm et al , 2000)

22

2. Growth evolution of the strain Burkholderia sacchari DSM 17165 under different carbon source combinations

An experiment was performed in order to optimize the growth media of the novel strain Burkholderia sacchari DSM 17165.

2.1 Materials and Methods

The method used by our working group consists in revitalizing existing colonies of the strain Burkholderia sacchari DSM17165 and reinoculate it on new plates with a known compatible media for the strain. The next day the flasks were inoculated, from the plates in a pre-culture media, in 300 ml shaken flasks( 100 ml media volume) for 24h at 37ºC. After around 12 to 18 hours the culture flasks were inoculated each with 10 ml from the pre-culture fermentations, the media used for both pre-culture and culture is shown in the following tables

Pre-cultures (LB) :

g/l To Weigh (g) Tryptone 15 7.5 Yeast extract 5 I) 2.5 NaCl 5 2.5 pH = 7 ( ) Total Volume (L) 0.5

Table 1. Compounds, respective concentration and mass weighed to prepare the pre-culture media

23

Culture media :

To Weigh g/l (g)

Na 2HPO 4.2H 2O 9 7.2

I) KH 2PO 4 3 2.4 pH = 7 ( ) (NH ) SO4 2 1.6 II) 4 2 MgSO 4.7H 2O 0.8 0.64

CaCl 2.2H 2O 0.02 0.016

III) NH 4Fe(III)Citrate 0.05 0.04 SL6 1.5 mL 1.2 mL Total Vol. (L) 0,8

Table 2. Compounds, respective concentration and mass weighed to prepare the fermentation media

To the described media the correct amount of carbon source must be added in order to obtain the final concentration of 15 g/l of sugar in each medium. SL6 is a solution containing some vital substances under residual concentrations.

The approach consisted on the parallel growth of colonies of B. sacchari DSM 17165 under different combinations of three carbon sources, sucrose, glucose and fructose. Seven different combinations of the previous carbon sources were made, resulting in seven distinct growth media. All the necessary compounds like potassium, nitrogen, phosphorus and other essential ions for the growth of the strain were added to the different media as previously shown. The different combinations of sugars can be seen on the next table.

Media description Sugar contents M1 Glucose M2 Fructose M3 Sucrose M4 Glucose + Fructose M5 Glucose + Sucrose M6 Fructose + Sucrose M7 Glucose + Fructose + Sucrose

Table 3. Media identification according to the sugars used

24 All three sugars added to each flask were in the same proportion, which means that in a flask with 2 sugars each sugar represents 50% of the total carbon source mass , and with 3 sugars each sugar represents 33% of the total carbon source mass.

The experiment here described was performed in 1 liter shaken flasks (250 ml media volume), and each medium was prepared in double which means the shaken flasks involved in the experiment totalizes 14. The adopted nomenclature for the flaks was M11 and M12 for the flasks containing the glucose only , M21 and M22 for the flasks containing only Fructose and so on.

The experiment was performed under sterile conditions and some samples were taken from each flask at regular intervals of time. The optical density was measured using a spectrofotometer at 420 nm, each sample was measured directly after collected, diluted if needed to give a correct OD reading value. The pH and ocasionally the glucose concentration was also measured from those samples, using quick test kits. In paralell 2 preweighed and labeled glass tubes were filled with 5 ml each from the reactor media.The tubes were centrifuged for 10 minutes at 5000 rpm. The supernatant was then collected in a plastic tube and frozen, the same happened to the labeled glass tubes that were previously centrifuged containing the pellets. This procedure was repeated for each sample collected at a designated time. In the end of the experiment the set of plastic tubes containing the supernatant from the samples was submitted to ammonia measurement and HPLC analysis, ( after defrozen). The set of glass tubes containing the pellets was liophilized, while still frozen, for aproximatelly 2 days, and then excicated for another day. The cell dry mass was then obtained by making the difference between the empty tube weight (tare) and the dry weight of the tube. The 3HB was extracted using a method similar to the one discribed by Steinbüchel et al, 1990. The polymer was submitted to a transterification and analysed in a gas chromatography device. The sugar concentration was analysed by submitting samples of the supernatant to a HPLC analysis. The ammonia contents was measured by measuring the conductivity of the supernatant samples, and then comparing the obtained values with the conductivity values obtained for standart solutions.

2.2.Results

When working with this strain as well as some others, in media where fatty compounds, like oleate, dodecanoate or others are not present, compounds that are known to seriously influence the optical density (O.D) readings in the spectrofotometer, it is common to apply an useful rule, the O.D reading value obtained under 420 nm divided by 6 will roughly represent the true cell

25 concentration within the sample in grams per liter (g/l). This way, by measuring the O.D. of the samples, which is a quick and simple procedure, it is possible to have an idea of the cell concentration within the sample at the designated time, and with that data make important decisions.

Evolution of Sugars and Ammonia Conc. on Flask M11 Evolution of Sugars and Ammonia Conc. on Flask M12

16 16 14 14 12 12 10 Glucose 10 Glucose 8 Total Conc. 8 Total Conc. 6 Ammonia 6 Ammonia Conc. (g/L) Conc.(g/L) 4 4 2 2 0 0 0 4 7 10 13 16 19 0 4 7 10 13 16 19 Time (h) Time (h)

Evolution of Sugars and Ammonia Conc. on Flask M21 Evolution of Sugars and Ammonia Conc. on Flask M22

16 16 14 14 12 12 10 Fructose 10 Fructose 8 Total Conc. 8 Total Conc. 6 Ammonia 6 Ammonia Conc.(g/L) Conc.(g/L) 4 4 2 2 0 0 0 4 7 10 13 16 19 0 4 7 10 13 16 19 Time (h) Time (h)

Evolution of Sugars and Ammonia Conc. on Flask M31 Evolution of Sugars and Ammonia Conc. on Flask M32

18 18 16 16 14 Sucrose 14 Sucrose 12 Glucose 12 Glucose 10 10 Fructose Fructose 8 8 6 Total Conc. 6 Total Conc. Conc.(g/L) Conc.(g/L) 4 Ammonia 4 Ammonia 2 2 0 0 0 4 7 10 13 16 19 0 4 7 10 13 16 19 Time (h) Time (h)

Evolution of Sugars and Ammonia Conc. on Flask M41 Evolution of Sugars and Ammonia Conc. on Flask M42

16 16 14 14 12 12 Glucose Glucose 10 10 Fructose Fructose 8 8 Total Conc. Total Conc. 6 6 Conc.(g/L) Conc.(g/L) Ammonia 4 Ammonia 4 2 2 0 0 0 4 7 10 13 16 19 0 4 7 10 13 16 19 Time (h) Time (h)

26 Evolution of Sugars and Ammonia Conc. on Flask M51 Evolution of Sugars and Ammonia Conc. on Flask M52

18 18 16 16 14 14 Sucrose Sucrose 12 12 Glucose 10 Glucose 10 Fructose 8 Fructose 8 6 6 Total Conc. Conc.(g/L)

Conc.(g/L) Total Conc. 4 4 Ammonia Ammonia 2 2 0 0 0 4 7 10 13 16 19 0 4 7 10 13 16 19 Time (h) Time (h)

Evolution of Sugars and Ammonia Conc. on Flask M61 Evolution of Sugars and Ammonia Conc. on Flask M62

18 18 16 16 14 Sucrose 14 Sucrose 12 12 Glucose Glucose 10 10 Fructose Fructose 8 8 6 Total Conc. 6 Total Conc. Conc.(g/L) Conc.(g/L) 4 Ammonia 4 Ammonia 2 2 0 0 0 4 7 10 13 16 19 0 4 7 10 13 16 19 Time (h) Time (h)

Evolution of Sugars and Ammonia Conc. on Flask M71 Evolution of Sugars and Ammonia Conc. on Flask M72

18 18 16 16 14 Sucrose 14 Sucrose 12 Glucose 12 Glucose 10 10 Fructose Fructose 8 8 6 Total Conc. 6 Total Conc. Conc.(g/L) Conc.(g/L) 4 Ammonia 4 Ammonia 2 2 0 0 0 4 7 10 13 16 19 0 4 7 10 13 16 19 Time (h) Time (h)

Graphic 1. Set of graphics that show the evolution of sugars and ammonia concentration for the 14 individual fermentations

The sugar concentration as well as ammonia concentration for each fermentation is shown on the above graphics, It is clear that after some time all the nitrogen (ammonia) and carbon sources (sugars) were completely consumed, the existence of both glucose and fructose in flasks M31 and M32 is explained by the hydrolysis of the sucrose, a diose, whose molecule is composed by a glucose unit plus a fructose unit. Naturally the hydrolysis of such compound results on the formation of glucose and fructose. Keeping in mind that “total concentration” is simply the sum of the concentration of the existent sugars on a specific period of time.

The combination of the data previously presented with the products of the fermentations will clarify some of the behaviour observed, as well as the final conclusion of which (among the tested) is the best sugar combination for the growth of the strain under study .

27 Time curves of CDM residual biomass and PHA Time curves of CDM residual biomass and PHA concentration on M12 concentration on M11 8 8 7 7 6 6 5 3HB 5 3HB 4 4 CDM CDM 3 3 residual biomass

residual biomass Conc. (g/L)

Conc. (g/L) 2 2 1 1 0 0 0 4 7 10 13 16 19 0 4 7 10 13 16 19 Time (h) Time (h)

Time curves of CDM residual biomass and PHA Time curves of CDM residual biomass and PHA concentration on M22 concentration on M21

8 7 7 6 6 5 3HB 5 3HB 4 CDM 4 CDM 3 3 residual biomass residual biomass Conc.(g/L)

Conc. (g/L) 2 2 1 1 0 0 0 4 7 10 13 16 19 0 4 7 10 13 16 19 Time (h) Time (h)

Time curves of CDM residual biomass and PH A Time curves of CDM residual biomass and PHA concentration on M32 concentration on M31

10 10 8 8 3HB 3HB 6 6 CDM CDM 4 4 residual biomass Conc. (g/L) Conc.

Conc.(g/L) residual biomass 2 2 0 0 0 4 7 10 13 16 19 0 4 7 10 13 16 19 Time (h) Time (h)

Time curves of CDM residual biomass and PHA Time curves of CDM residual biomass and PHA concentration on M41 concentration on M42

8 8 7 7 6 6 5 3HB 3HB

(g/L) 5 4 CDM 4 CDM 3 residual biomass 3 Conc.(g/L)

Conc. residual biomass 2 2 1 1 0 0 0 4 7 10 13 16 19 0 4 7 10 13 16 19 Time (h) Time (h)

Time curves of CDM residual biomass and PHA Time curves of CDM residual biomass and PHA concentration on M51 concentration on M52

8 8 7 7 6 6 5 3HB 5 3HB 4 CDM (g/L) . 4 CDM 3 3

residual biomass Conc residual biomass Conc.(g/L) 2 2 1 1 0 0 0 4 7 10 13 16 19 0 4 7 10 13 16 19 Time (h) Time (h)

28 Time curves of CDM residual biomass and PHA Time curves of CDM residual biomass and PHA concentration on M61 concentration on M62

8 8 7 7 6 6 3HB 5 3HB 5 CDM 4 CDM 4 residual biomass 3 residual biomass

3 Conc.(g/L)

Conc.(g/L) 2 2 1 1 0 0 0 4 7 10 13 16 19 0 4 7 10 13 16 19 Time (h) Time (h)

Time curves of CDM residual biomass and PHA Time curves of CDM residual biomass and PHA concentration on M71 concentration on M72

8 8 7 7 6 6 3HB 3HB 5 5 CDM CDM c. (g/L) c. 4 4 3 residual biomass residual biomass

Conc.(g/L) 3 Con 2 2 1 1 0 0 0 4 7 10 13 16 19 0 4 7 10 13 16 19 Time (h) Time (h)

Graphic 2. Set of graphics that show the time curves of cell dry mass, residual biomass and PHA concentration for the 14 individual fermentations

The careful combined analysis of both ammonia depletion rate as well as the residual biomass (the fraction of the cell dry mass composed by organelles, proteins and DNA, excluding the biopolymer contents), shows that as soon as the ammonia is depleted the cells stop growing in number and the verified increase on the cell dry mass is due to the “fattening” of the cells in biopolymer as previously explained. Except for the first media ( flasks M11 and M22 ) where the amonia was depleted at around 13 hours, in all the other media the depletion happened before 10 hours, which is in agreement with the stabilization of the residual biomass concentration and the rapid increase on the 3HB concentration. The stabilization of the 3HB near the end of the observed time is due to the depletion of carbon source, and knowing that carbon is the main constituent, in most strains the inexistence of this compound will block the biopolymer production.

The final decision on which should be the best combination for further experiments with the strain B. sacchari DSM 17165 must include a series of factors such as growth kinetics, polymer maximum concentration and naturally the carbon source costs.

29 The resumé of the fundamental data for the upcoming decision is shown in the following table

Media M1 M2 M3 M4 M5 M6 M7 µ (h-1) 0.60 0.69 0.71 0.62 0.79 0.74 0.59 Final C.D.M (g/l) 6.41 6.75 7.62 6.88 7.20 7.38 7.30 Final PHB (g/l) 2.27 2.71 3.47 2.84 2.91 3.01 3.05 Final % PHB 35.40 39.83 45.45 41.21 40.40 40.84 41.74

Table 4. Average calculations of specific growth rate, final cell dry mass concentration, final PHB concentration and final PHB percentage of the 7 different media

The information collected on the table was calculated as an average of both flasks corresponding to each medium, and taking into consideration that the deviation of the values correspondent to each flask from the calculated average was not significant.

2.3.Conclusions

In the end of the experiment, the medium 3 (sucrose only) proved to be the medium that suits better for fermentations using this B. sacchari strain. All the measured parameters like final cell dry mass, final PHB concentration and final PHB percentage, were maximized when sucrose was used as unique carbon source. Only the growth rate seems to be better when using glucose and sucrose simultaneously. The final decision should be adopting sucrose as the carbon source for future experiments envolving this strain.

The final conclusion can be partially explained by the fact that the stocks of the strain B. sacchari DSM 17165 that were used to perform this experiment were carefully selected during a series of generations, in order to be the best adapted strain to feed on an increased concentration of sucrose at 37ºC, this procedure was performed by cultivating the original strain on plates containing solely sucrose as carbon source under 37ºC, and by transferring the first colonies to develop into new plates with an increased sucrose concentration, by repeating this procedure during enough generations was possible to achieve a population of B.sacchari DSM 17165 that was able to quickly develop under 37ºC and a concentration of sucrose of circa 15 g/l. The high temperature needed (37ºC) can be explained by the fact that one of the partners of the working group was an enterprise operating in Brazil, and for a further scale-up using this bacteria it would be much more comfortable to them, economically speaking if they could operate with this strain at 37ºC, because in a tropical country cooling down a reactor to lower temperatures can be a seriously limiting issue. The increase on the carbon source concentration can be explained by the need to operate with bigger concentrations of carbon source when operating on batch-mode, and

30 naturally the carbon source if present on a concentration higher than a defined set point can be inhibiting for the bacteria growth, so, many times, the strain must be selected in order to develop properly under the desired parameters.

In spite of the possible reason explained above, the result was unexpected because sucrose, a diose, can not be immediately incorporated by the strain. Glucose enters almost immediately and fructose should enter immediately in the bacterial metabolic pathways, so it was expected that even the strain selected to grow better on sucrose would show better results on some of the other carbon source combinations.

Further experiments should now be performed in order to optimize the sucrose concentration as well as the concentration of other key factors such as nitrogen source, potassium and phosphorus concentration, etc. Also the strain should be studied to verify the existence or not of a secreted enzyme that breaks down sucrose, such an enzyme could have numerous applications both in the polymer production as well as other areas of biotechnology.

31 3.Determination of the growth kinetics of both strains Pseudomonas oleovorans ATCC 29347 and Pseudomonas putida KT 2442 under different fatty acids

The following experiment was performed with the medium chain polymer producer strains P. oleovorans ATCC 29347 and P. putida KT 2442 in order to check the behaviour of these strains when grown under different substrate conditions. It is known that the strains that produce medium chain polymers need to incorporate special fatty acid co-substrates in order to produce different co- polymers, the problem is that these needed substrates are usually highly toxic for the cells itself, deeply inhibiting the growth of the bacteria. Three different substrates were tested varying in availability, cost and purity, those were , oleic acid , a 18 carbon saturated fatty acid, decanoic acid, a 10 carbon saturated fatty acid, and saponified olive oil , the saponification process will not be explained in the present work, but is composed of a 2 step reaction and is the base for the soap industry. This saponified oil contains a mixture of fatty acids being the most representative the levulinic acid (5 carbon fatty acid). The comparison between both strains will not be executed due to the lack of a proper analysis that will be clarified later. They are presented together only because both experiments have been run in parallel and the initial growing media were the same for both strains.

3.1.Materials and methods

The strains were both grown under the same conditions and for each strain 3 different media were prepared all following the same basic recipe, only the diferrent co-substrates were added respectively. The media for both culture and pre-culture was as presented on the following tables

Pre-cultures:

g/L To Weigh (g)

Na 2HPO 4.2H 2O 4.5 2.25

I) KH2PO 4 1.5 0.75 pH = 7 ( ) (NH ) SO4 2.5 1.75 II) 4 2 MgSO 4.7H 2O 0.5 0.25

CaCl 2.2H 2O 0.02 0.01

III) NH 4Fe(III)Citrate 0.05 0.025 SL6 2 mL 1 mL IV) Glucose 15 7.5 Total Vol. (L) 0,5 Table 5. Compounds, respective concentration and mass weighed to prepare the pre culture media

32 Culture media

g/L To Weigh (g) 9 9 Na 2HPO 4.2H 2O I) 3 3 KH 2PO 4 pH = 7 ( ) (NH ) SO4 2.5 2.5 II) 4 2 1 1 MgSO 4.7H 2O 0.02 0.02 CaCl 2.2H 2O III) 0.05 0.05 NH 4Fe(III)Citrate SL6 2.5 mL 2.5 mL IV) Glucose 7 7 Total Vol. (L) 1

Table 6. Compounds, respective concentration and mass weighed to prepare the fermentation media

Each of the numbers I , II, II and IV represents what was added to each flask prior to autoclaving, in order to avoid crossed reactions and the formation of precipitates due to the high temperature and pressure. After the autoclaving and cooling down all the 4 parts of the media were blended together in one flask. To each of the different 3 media the needed amount of the different fatty acids was added in order to achieve the desired concentration of 3 g/l . Each fermentation was performed in double and to each shaken flask was attributed a number and a letter as shown in the following tables Strain P. oleovorans ATCC 29347

Media description Fatty acid contents A1 Oleate A2 B1 SOO (saponified olive oil) B2 C1 Dodecanoate C2

Strain P. putida KT 2442

Media description Fatty acid contents A3 Oleate A4 B3 SOO (saponified olive oil) B4 C3 Dodecanoate C4

Table 7 and 8. Media nomenclature and respective fatty acid contents for each individual fermentation

33

All the analysis methods were similar to ones discribed in the previous experiment, except for the polymer isolation that was not performed.

3.2.Results

The information resulting from this experiment will be presented in 2 sets of graphics, the first containing the residual carbon source and ammonia concentrations and the second the optical density and cell dry mass. The lack of detailed information about the biopolymer produced inviabilized further analysis on the polymer itself, since there were not available standards of the monomers constituent of the medium chain polymer produced to perform the gas chromatography analysis.

Glucose and Ammonia concentrations in A2 Glucose and Ammonia concentrations in A1

7 7 6 6 5 5 4 Glucose 4 Glucose 3 NH4+ 3 NH4+ Conc. (g/L)

Conc. (g/L) 2 2 1 1 0 0 0 10 20 30 0 10 20 30 Time (h) Time (h)

Glucose and Ammonia concentrations in B2 Glucose and Ammonia concentrations in B1

7 7 6 6 5 5 4 Glucose 4 Glucose NH4+ 3 NH4+ 3 Conc. (g/L) Conc. (g/L) 2 2 1 1 0 0 0 10 20 30 0 10 20 30 Time (h) Time (h)

34 Glucose and Ammonia concentrations in C1 Glucose and Ammonia concentrations in C2

7 7 6 6 5 5 4 Glucose 4 Glucose 3 NH4+ 3 NH4+

Conc.(g/L) 2 Conc.(g/L) 2 1 1 0 0 0 10 20 30 0 10 20 30 Time (h) Time (h)

Graphic 3. Set of graphics that show glucose and ammonia concentrations for the fermentations performed by the strain P. oleovorans ATCC 29347

Glucose and Ammonia concentrations in A4 Glucose and Ammonia concentrations in A3

8 8 7 7 6 6 5 5 Glucose Glucose 4 4 NH4+ NH4+ 3 3 Add. Glucose Add. Glucose

Conc.(g/L) 2

Conc. (g/L) 2 1 1 0 0 -1 0 10 20 30 40 50 -1 0 10 20 30 40 50 Time (h) Time (h)

Glucose and Ammonia concentrations in B3 Glucose and Ammonia concentrations in B4

8 8 7 7 6 6 5 5 Glucose Glucose 4 4 NH4+ NH4+ 3 3 Add. Glucose Add. Glucose Conc.(g/L) 2 Conc. (g/L) 2 1 1 0 0 -1 0 10 20 30 40 50 -1 0 10 20 30 40 50 Time (h) Time (h)

Glucose and Ammonia concentrations in C3 Glucose and Ammonia concentrations in C4

9 9 8 8 7 7 6 Glucose 6 Glucose 5 5 NH4+ NH4+ 4 4 3 Add. Glucose 3 Add. Glucose Conc.(g/L) Conc.(g/L) 2 2 1 1 0 0 0 10 20 30 40 50 0 10 20 30 40 50 Time (h) Time (h)

Graphic 4. Set of graphics that show glucose and ammonia concentrations for the fermentations performed by the strain P. putida KT 2442

35

Note that the duration of the fermentations with the different strains is also different. The experiment with the strain P. oleovorans ATCC 29347 lasted for 25 hours, while the experiment with the strain P. putida KT 2442 lasted for 43 hours and was subjected to a re-feed of glucose of 7 g/l in each flask.

Optical Density for the different fermentations

30

25

A1 20 A2 B1 15 B2 C1 O.D. (420nm) 10 C2

5

0 0 15 20 25 Time (h)

Graphic 5. Optical density measured in each fermentation for the strain P. oleovorans ATCC 29347

36 Cell Dry Mass concentration for the different fermentations

4,5

4

3,5 A1 3 A2 2,5 B1

2 B2

C.D.M. (g/L) C1 1,5 C2 1

0,5

0 0 15 20 25 Time (h)

Graphic 6. Cell dry mass and measured in each fermentation for the strain P. oleovorans ATCC 29347

Optical Density for the different fermentations

30

25

A3 20 A4 B3 15 B4 C3 O.D. (420nm) 10 C4

5

0 0 15 20 24 43 Time (h)

Graphic 7. Optical density measured in each fermentation for the strain P. putida KT 2442

37 Cell Dry Mass concentration for the different fermentations

6

5 A3 4 A4 B3 3 B4 C3

C.D.M. (g/L) C4 2

1

0 0 15 20 24 43 Time (h)

Graphic 8. Cell dry mass and measured in each fermentation for the strain P. putida KT2442

It is clear from the combined analysis of the O.D and CDM that in the present case the empirical relation between OD and CDM explained in the previous experiment is not valid. This is due to the fact that the presence of fatty acids within the samples taken during time highly influenciate the O.D. value obtained, therefore the O.D reading is more an indicator of the presence of the fatty acid than the cell concentration itself.

38 3.3 Conclusions

Taking into account that the only variable among the experiments for each strain is the acid used, the growing behaviour can now be analysed independently for each strain.

For the strain P. oleovorans ATCC 29347 all the available glucose was consumed in the first 15 hours as well as the ammonia source available. The obtained final cell dry mass as well as the growth behaviour is very similar for the 3 media, being the results achieved on the media containing dodecanoate slightly worse in terms of final cell dry mass. Adding the fact that dodecanoate is, from the 3 fatty acids used, the one that has a higher cost, this precursor should not be used if the objective is to achieve high cell dry mass concentrations. Comparing the SOO and oleate as co-substrates the insignificant differences in the general behaviour of the cultures should make the choice rest upon the SOO since it is directly obtained form the olive oil making it the most acessible of the 3 tested fatty acids.

The strain P. putida KT 2442 shows a much more diversified behaviour than the strain P. oleovorans ATCC 29347 . The strain seems to be really sensible to the toxicity of the dodecanoate, result shown by the extremely slow growth when compared to the other substrates and by the fact that both carbon and ammonia sources were never depleted during the entire experiment. Comparing the behaviour of the strain when fed with the 2 remaining substrates, the carbon and ammonia source presented a similar consumption rate for both cases, although the final cell dry mass and growth behaviour was slightly better for the oleate. The choice of the best substrate for this strain should really depend on the size of the experiment or future industrial production. For smaller scales it should be used the oleate in order to obtain slightly better cell dry mass concentrations, on a bigger scale the reduced price of SOO would most likely overtake the difference in cell dry mass concentration.

Unfortunately for the explained reasons above it was not possible to perform a gas chromatography analysis that would give the values of the obtained polymer concentrations for each case, and possibly completely different conclusions.

Further experiments would be much more interesting if the polymer concentration and composition could be determined, if that was the case after defining the best substrate, the optimal concentration of precursor , in order to avoid any toxic effect and produce the desired co-polymers, could be achieved. The strains could be selected as done with Burkholderia sacchari in order to obtain a strain that grows properly under higher fatty acid concentrations, as well as try new saponificated oils, like sunflower oil, that has a considerably lower cost than olive oil and is readily available, or other oils from the many existent.

39 4. Growth behaviour at a Bioreactor scale of the Pseudomonas oleovorans ATCC 29347 and Pseudomonas putida KT 2442 using the precursors oleate and dodecanoate.

The scl-PHA ( short chain length PHA) are at this point exhaustively studied. Scientists are now able to obtain extremely high concentrations of polymer (above 90% of the cell dry mass) with some of the scl-PHA producer strains. On the other hand the mcl-PHA (medium chain length PHA) are still not well studied, and the yields obtained with the strains that produce this kind of polymer are still relatively low when compared to the scl-PHA producers.

The objective of this experiment was, by performing a scale up of the previously executed experiment, try to enhance the yields of both biomass and polymer production. The bio-reactor scale in fact cannot even be compared to the shaken flask scale, both the agitation and oxygen feed are completely different, any result obtained under a shaken flask scale should be expected to improve under a bioreactor scale.

4.1.Materials and Methods

. For this experiment the mcl-PHA producers P. oleovorans ATCC 29347 and P. putida KT 2442 were used.

A set of 2 stirred-tank-reactors of 1.5 l volume was prepared each one to house one of the strains.

The inoculation procedure consisted of using plates where the strains were grown for one day, on the second inoculate 300 ml shaken flaks with these plates, on the third day inoculate 1 liter shaken flasks with 10 ml of the 300 ml culture, and finally on the forth day inoculate the reactors with 1 liter flasks culture.

The pre-culture media used in the 300 ml and 1 liter flasks, as well as the culture media used for both reactors are shown on the following tables

40

Pre-cultures and Inoculum:

g/L Na HPO .2H O 9 I) 2 4 2 KH 2PO 4 3 (NH ) SO4 2 II) 4 2 MgSO 4.7H 2O 0.4

CaCl 2.2H 2O 0.02 III) NH 4Fe(III)Citrate 0.05 SL6 2 mL IV) Glucose 15 Total Vol. (L) 3

Table 9. Compounds and respective concentration used to prepare the pre culture and inoculum media

React ors A and B

g/L I) Na 2HPO 4.2H 2O 5

(NH 4)2SO4 2.5 II) MgSO 4.7H 2O 0.5 NaCl 1

CaCl 2.2H 2O 0.02 III) NH 4Fe(III)Citrate 0.05 SL6 3 mL IV) Glucose 20 Total Vol. (L) 1+0.5 (INOC.)

Table 10. Compounds and respective concentration used to prepare Reactor A and B media

Some solutions were prepared to add a posteriori to both reactors, a solution of glucose was prepared to re- feed the reactors, solutions of oleate to feed both reactors during the “fattening” phase, a solution of dodecanoate to feed only the strain P. oleovorans ATCC 29347, the high toxicity of this compound to the strain P. putida KT 2442 was already identified during the previous experiment. Solutions of diluted sulphuric acid as well as diluted NaOH to correct the pH if needed, and finally a solution of NH 4OH used to both raise the pH during the growing phase and to feed ammonia (nitrogen) to the system. When the system was switched from growing to fattening phase the used base was also switched from NH 4OH to NaOH, because the trigger to the PHA accumulation was in the present case the lack of nitrogen source.

41

The side solutions prepared were as follows:

Carbon Source & Precursors :

2 x Glucose 50% Solution (0.5Kg Glucose + 0.5Kg H2O) Decanoate 10% m/m (225g H2O+25g deca.) 2 x Oleate 10% m/m (450g H2O+50g olea.)

ACID - 2x250mL H2SO4 10% solution BASE - 2x250mL NaOH 10% solution + 2x500mL NH4OH

The fermentations were conducted during a variable span of time. These strains are actually extremely sensible to the pH within the fermentation media, they grow at an optimum pH of 7 , any pH value slightly different, whether is more acid or more alkaline will drive the bacteria into stress making them to produce huge amounts of foam, that foam can be controlled with antifoam during a small period, but if the pH doesn’t return quickly to the optimum value, foam will be continuously produced, and the culture will soon be flooded with anti-foam (a greasy compound that inhibits bacteria if present on an excessive concentration).

During the preparation of one of the reactors, reactor 4, that would house the strain P.oleovorans ATCC 29347, the pH probe was malfunctioning. Without a spare probe, the group decided to still carry on the experience on that reactor, trying to control the pH manually by taking samples and measuring the pH and act accordingly to the pH reading on the samples. The experiments started but due to the quick dynamics of the fermentation soon it was impossible to keep the pH under control on reactor 4, so the experiment on that reactor was finished before the needed time to obtain the desired data due to an excess of foam produced that corrupted the air filter and by doing that contaminated the entire fermentation.

Once again due to the problem described above the comparison between the results on both strains can only be done until a certain degree, and the massive behaviour differences shown by the previous experiment also corroborates the same statement.

All the analysis methods were similar to ones discribed in the previous experiment, except for the polymer isolation that was not performed.

42 4.2.Results

The data collected for reactor 3 ( P. putida KT 2442 ) will now be presented and analysed. The cell dry mass concentration and optical density over time can be seen on graphic 9.

CDM vs OD for reactor 3

30 250

25 200

20 150 CDM OD 15

CDM (g/L) 100 OD (420nm ) 10

50 5

0 0 0 3 6,25 8,5 10 12 14 16 18 20 23 26,5 29,5 33,5 36,5 39,35 Time (h)

Graphic 9. Cell dry mass concentration and optical density values observed on reactor 3

The glucose concentration over time as well as the glucose and oleate additions performed can be seen on graphic 10.

Glucose concentration and glucose and oleate additions over time

18

16

14 Glucose 12

Glucose 10 Addition 8 Oleate Conc. (g/L) Addition 6

4

2

0 0 5 10 15 20 25 30 35 40 Time (h)

Graphic 10. Glucose concentration, glucose and oleate additions over time for reactor 3

43

Finally , the ammonia concentration over time is presented on graphic 11

2

1,8

1,6

1,4

1,2 NH4+ 1 Time of Nitrogen Source Feed

Conc. (g/L) 0,8 Stoped

0,6

0,4

0,2

0 0 5 10 15 20 25 30 35 40 Time (h)

Graphic 11. Ammonia concentration over time on the reactor 3

In the table 11 are shown all the additions done to reactor 3

<<< Additions >>> Added Sum added Added Oleate Time (h) Glucose (g/l) Glucose (g/l) NH4OH (g) (g/l) 6 2.5 2,5 7 2.5 5 8 5 10 9,5 5 15 10,5 10 25 12,5 5 30 13,75 5 35 15 10 45 17 10 55 18,8 10 65 21,8 65 2 30,5 2.5 67.5 35 2.5 70 19

Table 11. Glucose, ammonia hydroxyde and oleate additions performed on reactor 3

By observing the ammonia concentration over time it is clear that after the end of the nitrogen source feed the ammonia concentration dropped a little until the next sample which is a sign that the biomass was still being formed until around 22 hours from the start, approximately the same time that the oleate was added. After that it is clear by the increase in the ammonia concentration in the media that the biomass was dying, most likely due to the toxic effect of the

44 oleate. Observing the CDM concentration it is not clear, because part of the biomass was dying, but probably the culture was also accumulating PHA, so by the direct reading of the CDM values it is not clear the toxic effect of the oleate that is also a fundamental substrate for the polymer production. The OD peak seen at around 30 hours can be due to a handling problem while measuring the OD on that sample, because samples that contain fatty acids are usually extremely hard to handle because the oleate is less dense than the rest of the media and does not dilute properly.

A more complex analysis would require the measurement of the PHA concentration under gas chromatography, unfortunately as the standards needed to do so were not available that analysis was not performed as previously explained.

The data from reactor 4, where the strain P. oleovorans ATCC 29347 was grown, will be displayed in the next series of graphics

CDM vs OD for reactor 4

30 50

25 40

20 30 CDM OD 15

CDM (g/L) 20 OD (420nm ) 10

10 5

0 0 0 3 6,25 8,5 10 12 14 16 18 20 Time (h)

Graphic 12. Cell dry mass concentration and optical density values observed on reactor 4

45 The glucose concentration over time as well as the glucose and oleate additions performed can be seen on graphic 13

Glucose concentration and glucose and oleate aditions overtime

45

40

35 Glucose 30

25 Glucose Addition 20 Oleate Conc. (g/L) Addition 15

10

5

0 0 5 10 15 20 Time (h)

Graphic 13. Glucose concentration, glucose and oleate additions over time for reactor 4

Again, finally the ammonia concentration over time is presented on graphic 14

Nitrogen cocentration and aditions overtime

2

1,8 NH4+

1,6

1,4 Time of Nitrogen Source Feed Stoped 1,2

1 NH4+ Addition

Conc.0,8 (g/L)

0,6

0,4

0,2

0 0 5 10 15 20 Time (h)

Graphic 14. Ammonia concentration over time on the reactor 4

46 In the table 12 are shown all the additions done to reactor 4

<<< Additions >>> Added Glucose Sum added Added NH4OH Time (h) Oleate (g/L) (g/L) Glucose (g/L) 3,5 3.33 4 2.67 5,5 2.5 2.5 7 5 7.5 8 5 12.5 9,5 5 17.5 3.33 10,5 10 27.5 12,5 5 32.5 13,75 2.5 35 18,7 5 40 2.67 21,8 2

Table 12. Glucose, ammonia hydroxyde and oleate additions performed on reactor 4

Taking into consideration that the strain P. oleovorans ATCC 29347 is highly sensible to pH variations, and as previously said the pH probe was not working, it was really difficult to keep up the pH value overtime. Therefore and as the pH was near 7 untill the 10 th hour from start, the biomass growth was good, what can be seen by the CDM and OD, note that at this point no oleate or other fatty compound has been added, so the OD reading gives an accurate indirect value of the biomass concentration, this conclusion is corroborated by the low glucose and nitrogen concentration until this point. From the 10 th hour on problems in keeping the pH under the desired values started to appear. Nevertheless the CDM was kept at around 5 g/l until the point where the oleate was added at 22 hours from start when the pH rose drastically due to the alkalinity of the oleate solution ( pH over 10), the biomass inside the reactor started foaming heavily, the air filter got corrupted and the experiment was immediately finished by killing the biomass by rising the reactor temperature to 70ºC for a period of time. The dodecanoate was never used, it was supposed to be added to the reactor 4 under low concentrations in parallel with the oleate additions. Unfortunately with all the problems that occurred the experiment was finished before there was the possibility to add this precursor.

47 4.3.Conclusions

As previously stated, oleate is toxic to the mcl-PHA producers such as P. putida KT2442, the strain used in the present experiment. The maximum biomass concentration achieved (27 g/l), prior to the oleate addition, lowers immediatelly and continuously when oleate is added to the media. Apparentelly the the objective of producing PHA by using fatty acid precursors is being overtaken by the toxicity of the oleate, resulting on a loss of cell dry mass. Fact proven by the final cell dry mass concentration of 18 g/l. The solution should be a careful monitoring of the oleate concentration in order to keep it under 1 g/l as well as to prepare the strain to thrive under higher oleate concentrations with a similar method described previously for the strain B. sacchari DSM 17165 to thrive under high temperatures. Once again the experiment would only be complete with a complete analysis of the polymer produced, as well as the weight percentage of the cell dry mass in polymer. The experiment with strain P. oleovorans ATCC 29347 can be considered a failure, the results are exposed in this work as a proof that the strain is highly sensible to the pH variations, a pH value a slightly different from the optimal value (6.9), immediately causes problems, actually this parameter is much more important in the strain behaviour than any other of the controlled ones, like temperature, stirring speed at the concentration of any of the substrates.

48

Part 2 : Acid Hydrolysis for the Production of Fermentable Sugars from Waste Raw Materials

5.1 Introduction

One of the main concerns within the biopolymer industry is to achieve a competitive production cost. Nowadays the biopolymer industry is still not able to compete directly with the petrochemical polymer industry, in fact the production cost of a defined biopolymer is a few times higher than its petrochemical equivalent.

One of the approaches to the problem is to find a suitable carbon source for the production of the desired biopolymer which is readily available and above all, cheap. Taking into consideration that the industrial production of PHA’s is mediated by aerobic microorganisms, only a maximum of 50% of the total carbon source will effectively be transformed into biomass and polymer, the rest will be lost through the intracellular respiration. The utilization of waste materials upgraded to the role of starting material for the PHA biosynthesis on one side constitutes a cost-efficient strategy to overcome the carbon source expenses problem and on the other hand helps the industry to overcome their waste disposal problem.

The strategy utilized by our working group consisted on a 2 step experiment; the first one consisted on performing an acid hydrolysis in order to obtain fermentable sugars from a set of agricultural waste materials. The second step consisted on producing PHA from the obtained fermentable sugars by means of a fermentation using a suitable strain.

The chosen waste material for starters were two different types of “marc” ( waste material composed by the seeds, skins and pulp from the olive oil extraction), the first type that will be called “marc 1” is the waste obtained directly from the olive oil production. The second type that will be called “marc 2” is the waste obtained after the “marc 1” is processed and a secondary oil is removed. The third and last starting material was tomato waste obtained directly from the tomato pulp industry and it consists of the dry skin, seeds and also some dry pulp. In fact all these waste materials represent the solid fraction of the respective industries.

These waste materials are mainly composed by lignocellulosic materials (consisting of lignin, cellulosic and hemicellulosic fibres). The development of a suitable digestion of these lignocellulosic materials which means the effective breakdown of cellulose and hemicellulose into microbial convertible sugars (hexoxes and pentoses) are the prerequisites for an efficient biotechnological conversion of these promising raw materials into the desired end products.

49

According to the literature cellulose is constituted solely by a repetition of glucose units and hemicellulose is composed by a wide variety of both hexoses and pentoses being the most important constituent the xylose.

The kinetics of degradation of cellulose and hemicellulose are strictly connected to the degradation of glucose, xylose and other constituents, work was done in order to optimize the hydrolysis process, so a reasonable concentration of fermentable sugars could be achieved, that was done by optimizing both time of hydrolysis, temperature and acid concentration.

Three processes for the production of fermentable sugars from waste materials are widelly used.

The alkaline hydrolysis is similar to the acid hydrolysis but using bases instead of acids, although according to Nzelibe et al this process has much lower yields compared to acid hydrolysis.

The third process the enzymatic hydrolysis involves the usage of purified enzymes or special strains for the process, the results achieved are very good, but it is a complex, difficult and long process.

Therefore the acid hydrolysis was the procedure adopted for the desired objective.

5.1.1.Hemicellulose removal – Xylan removal

In the past scientists investigated the effect of acid concentration and temperature in the xylan removal rate from aspen wood. Xylan is the main component of hemicellulose. He found that the initial rate constant for the removal of xylan in this wood can be described by the following equation

log 10 (k/CH) = 15.083 - 6171.3/T + 0.22219(CH) Eq. 1 (Zerbe et al, 1985)

Where CH = molarity of hidrogen ion [H +], T = absolute temperature (°K) , k = rate constant (min –1).

Other scientists studied the same subject on other types of woods and found that the initial decomposition rate of xylan doesn’t vary significantly from the kinetics given by the equation above. It can be presumed that working with other material where xylan is present the same kinetic model should apply, therefore it will be considered valid for the experiments described in the present work.

50

Scientists observed that within a sample, after the percentage of xylan removed reached about 60%, the model here presented was no longer valid, in fact experiments showed that the rate of xylan removal can be described as the sum of two linear curves as shown in figure 9.

Figure 9. Graphic representation of the xylan removal curve as the result of the sum of two linear curves(Zerbe et al . 1985)

The combined curve can be described by the following equation

XR = xylan remaining in residue, % of original

= A 0exp( – k 1t) + B 0exp( – k 2t) Eq.2

where t = time.

Taking into consideration that if t=0

XR=A 0+B 0=100 Eq.3

The equation can be described as:

XR = A 0exp( – k 1t) + (100 – A 0)exp( – k 2t) Eq.4

The values of A 0, k 1 and k 2 can be determined by experimental data giving an idea of the xylan removal rate for a given temperature and acidity. Other carbohydrates can be found on hemicellulose, but their minimal concentration makes them not interesting for the present work and they will not be considered (Zerbe et al . 1985).

51

5.1.2. Xylose degradation

The breakdown of xylan, which is composed of xylose, mainly, gives origin to free xylose units. However the xylose itself degradates into the so called degradation productions being the most important the furfural

Figure 10. Schematic representation of the xylose degradation pathway (Zerbe et al 1985)

Although the furfural has a high commercial value, the objective of the present work is to maximize the xylan breakdown and minimize the xylose decomposition, in order to obtain fermentable sugars.

Figure 11. Typical chromatogram obtained from the HPLC analysis of a xylan hydrolysis sample (Zerbe et al 1985)

Data on the degradation rate of xylose, covering a broad range of temperatures, acidity and xylose concentration can be found on the work performed by Root 1956; Root et al . 1959. According to this author the kinetics of the degradation of xylose under acidic conditions can be described by the following equation (Zerbe et al, 1985)

k = 2.72 . α. δ. γ.CA·exp[-35.7(473.1-T)/T] Eq.5

52 α(CX)= a function of xylose molarity where CX= xylose molarity δ(T) = a function of temperature γ(T,CA)= a function of temperature and acid normality CA= acid normality T=absolute temperature (ºK)

No functional forms are available for α, δ, γ, but their relationships to the independent variables can be found in tabular form (Root 1956; Root et al , 1959).

5.1.3. Cellulose breakdown and glucose degradation

The cellulose hydrolysis rate as well as glucose degradation rate can be roughly described by the following equations

Eq. 6

Eq. 7

Where C A is the concentration of cellulose and C B is the concentration of fermentable sugars from cellulose, K l is the reaction rate constant for cellulose to sugar K 2 is the reaction rate constant for sugar to decomposition products t is time(Yan et al . 2008).

The value os both K l and K 2 can be calculated by the following equation

Eq. 8

The value of Pi depends on the used acid concentration, in figure 12 are some of the values depending on the acid concentration (Yan et al . 2008)

53

Figure 12 . Values for the constants P1 and P2 dependant on the acid concentration, values for the activation energies ∆H1 and ∆H2( Yan et al 2008)

The model presented above actually represents a simplified model of the complex kinetics envolved in free glucose formation and degradation as shown by figure 13.

Figure 13. Cellulose hydrolysis model (Zerbe et al, 1985)

The most important decomposition products of glucose is HMF (hydroxymethylfurfural), this compound when degradated gives origin to levulinic acid and formic acid. The HPLC analysis of the hydrolysates may show peaks corresponding to these compounds if the degradation of glucose happens in considerable amounts. The kinetics shown here for glucose breakdown correspond to the breakdown of the resistant cellulose, the readily hydrolysed cellullose follows a kinetic system much more similar to the hemicellulose.

54 5.1.4. Cellulose vs. hemicellulose breakdown

The combined analysis of hemicellulose and cellulose degradation kinetics is shown by the figures 14 and 15.

Figure 14. Graphic representation of the hemicellulose breakdown constant k, dependant on acid concentration, at 120ºC (Zerbe et al 1985)

Figure 15. Graphic representation of the cellulose breakdown constant k c, dependant on acid concentration, at 180ºC(Zerbe et al 1985)

Taking into account that the temperature at which the measurements were taken in the xylan hydrolysis is 120ºC and the temperature in the cellulose hydrolysis is 180ºC and the hydrolysis constants rise with the rising on the temperature, it is clear that the hydrolysis of the xylan is much faster that the cellulose hydrolysis for the same temperature and acid conditions, therefore the obtained xylose from the xylan is subjected to degradation much faster than the glucose, when hydrolysing all the material in a single step.

55 The best approach to avoid a high degree of xylose degradation is to proceed to hydrolysis in a two step system. The first that could be called pre-treatment could be performed by subjecting the raw materials to a set of conditions much less aggressive, with a lower temperature and lower acid concentration, in order to obtain a high xylose yield from xylan. Subsequently, the insoluble cellulose containing the greatest part of the glucose, which at this point is present within the solid part of the mixture, should be separated from the liquid fraction that would contain the free xylose as well as other compounds obtained from the pre-treatment procedure. The solid fraction should now be subjected to the second step the hydrolysis itself, under the much more agressive temperature and acid conditions in order to break down the cellulose matrix, in order to retrieve the free glucose for further fermentation.

Obviously the optimization of both steps would require a full understanding of the composition of the raw materials, the xylan/cellulose ratio as well as the presence of other nonrelated compounds as proteins and fatty acids.

As previously mentioned the objective of the present work is not to obtain the optimal carbohydrates yields, but simply reasonable yields in order to advance to the fermentation step. The scarcity of time and information related to the substrates composition, made the optimization of the process impossible, therefore only reasonable amounts of both xylose and glucose were obtain after a set of trials for the different substrates.

The analysis of complex raw materials, as the ones studied here, like feedstock and others, presents a serious challenge. The HPLC graphics obtained are usually confuse with overlapped peaks corresponding to different compounds, as well as a huge set of peaks for each sample. Therefore only the most signicant peaks will be taken into consideration, the ones related to the breakdown of cellulose and hemicellulose as well as their respective degradation products, as previously explained.

56 5.2. Acid hydrolysis for the production of fermentable sugars from complex raw materials

The hydrolysis were performed in a custom made steel reactor, capable of withstand the extreme conditions of high acidity combined with high pressure and temperature. The reactor was cylindrical with a volume of 800 ml. Plugged to it there were 2 taps in series, in order to obtain samples while performing reactions at high pressure. There were another 2 taps in the top of the reactor, where a plastic pipe was attached in order to deliver some N 2 inside the reactor to turn the atmosphere inert, at the beginning of the experiment. The other objective of these taps was also to release the vapours formed during the experiment, in the end of it. The reactor was on a heating/stirring plate, and naturally there was a magnet inside the reactor.

The temperature, as well as the stirring speed were controlled indirectly by the regulators on the heating plate.

In the beginning of the experiment procedure, Marc 1 was the raw material chosen to optimize the hydrolysis conditions, because it was the material we had in bigger amount and it was the cheapest of all 3 materials to be studied.

The first approach adopted consisted on submitting a 500 ml solution containing 5 g of raw material Marc 1 to the hydrolysis under the apparatus described above, with a solution of concentrated cloridric acid (6 M) and a temperature of around 90ºC.

The first results showed that the adopted conditions were not correct, both peaks of glucose and xylose were barely noticeable, while the fructose peak wasn’t even present. The concentrations obtained of the 3 sugars were residual, therefore the conditions had to be dramatically changed in order to improve the results.

57

Graphic 15. HPLC chromatogram of the first acid hydrolysis performed, 5 g of Marc 1, acid concentration of 6M, temperature of 90ºC

Only some representative samples are shown on the chromatograms, otherwise the graphics would be too overloaded and impossible to distinguish anything. Only 3 or 4 samples are shown in each graphic, the rest of the results are shown in the correspondent tables.

The huge peak obtained at around 6 minutes represents the NaCl present in the sample. The extremely acid samples were previously neutralized with NaOH to avoid damaging the HPLC column, the resulting salts in this case NaCl, is detected by the hplc, and naturally a high quantity of this salt was formed during the neutralization due to the high concentration of HCl in the sample. The peak at around 15 minutes is obtained due to an impurity present in the water used to run the HPLC, it will be present in all HPLC chromatograms, and it is completely irrelevant for the present study.

The second attempt consisted on keeping the acid conditions and raising the temperature to around 105 ºC, as well as raising the amount of Marc1 to 20g in 500 ml solution , in order to achieve more noticeable peaks and to lower the reading errors attached to the analysis of such a low quantity of raw material . The results were significantly better, which can be immediately realised by the respective HPLC chromatogram shown in graphic 13 and the table 13 .

58

Graphic 16. HPLC chromatogram of the second acid hydrolysis performed, 20 g of Marc 1, acid concentration of 6M, temperature of 105ºC, rose curve (Sample 1), blue curve (sample 3), black curve (Sample 5), brown curve (sample 7)

Fermentable Time ( min ) Glucose (g/l) Xylose (g/l) Fructose (g/l) sugars (g) / g raw material

Sample 0 0 0 0 0 0 Sample 1 15 0 1.67 0 0.04 Sample 2 30 0 1.14 0.49 0.04 Sample 3 40 0.46 0.46 0 0.02 Sample 4 60 0.91 0.45 0 0.03 Sample 5 80 1.07 0 0 0.03 Sample 6 120 1.14 0 0 0.03 Sample 7 180 1.33 0 0 0.03

Table 13. Glucose, Xylose and Fructose concentrations and Fermentable sugar yield for each sample taken in the second acid hydrolysis

59

By the analysis of the graphic and table it is clear that under the current conditions, an immediately high concentration of xylose was obtained (peak at around 11 minutes), with almost no glucose present (peak at 9,5 minutes), with the advance of the experiment, the xylose concentration started to decrease drastically, while the glucose concentration was getting higher, the fructose concentration (peak at 12,5 minutes) remained insignificant during the whole process. Something had to be changed if we wanted to preserve the xylose produced as well as achieve a high concentration of glucose.

At this point and due to inexperience, the trials were being conducted on a trial and error basis, some of the fundamentals as low acid concentration to be used, combined with high temperature and a 2 step process in order to preserve the xylose and fructose obtained and after maximize the glucose production were not known and therefore not used. The experience as well as a continuous increasing in the hydrolysis knowledge led to a more optimized process.

Some more hydrolysis were performed, being temperature and acidity tested in different combinations, the results quality increased with a diminishing in the acid concentration, having the temperature also an important but not so strong effect as the acid concentration, as previously showed by the kinetics equations.

Eventually a completely new approach was adopted, the samples were to be subjected to a pre-treatment of heating the raw material (15 g of Marc 1) solely in water (500 ml) for a determined period. It was determined that the apparatus would need approximately one hour to reach he desired temperature of about 160ºC, after that, leave the pre-treatment phase running for another 30 mins. After this initial step the apparatus needed to be cooled down quickly and the sulphuric acid added until a concentration of 50 mM was reached, and the apparatus would be sealed again and heated to 160ºC and samples taken at regular intervals. This procedure was based on the work of Mosier et al , 2002.

The results obtained were not immediately as expected, as can be seen by the results shown in graphic 17 and table 14.

60

Graphic 17. HPLC chromatogram of hydrolysis following the Mosier method , 15 g of Marc 1, acid concentration of 50 mM, temperature of 160ºC, black curve (Sample 2), rose curve (sample 3),blue curve (Sample 4)

Fermentable Time ( min ) Glucose (g/l) Xylose (g/l) Fructose (g/l) sugars (g) / g raw material

Sample 1 60 0 1.13 0.71 0.06 Sample 2 120 0.59 1.18 0.82 0.09 Sample 3 180 0.61 0.82 0.69 0.07 Sample 4 Day after 0 0 0 0

Table 14. Glucose, Xylose and Fructose concentrations and Fermentable sugar yield for each sample in the acid hydrolysis following the Mosier method

61

The best results were obtained with sample 2 taken after 120 minutes from start with a total concentration of 2.59 g/l of fermentable sugars, corresponding to a conversion of around 9% of the raw material, into fermentable sugars. The importance and continuous growth of the peaks at 29 and 40 minutes will be later explained.

As the results obtained with the Mosier method were not satisfactory enough, a method described by Saeman et al ,1945 , appeared to be promising.

Doubts started to appear regarding the quality of the raw material itself, there was the possibility that the material would be so poor in convertible sugars, that the problem would pose on the materials used themselves instead of the adopted methods. Therefore a quick experiment was performed by using the Saeman method on pure cellulose, the results shown on graphic 18 were obtained by using a sulphuric acid concentration of. 100 mM and a temperature of 180 ºC with 500ml of solution and 10g of cellulose.

Graphic 18. HPLC chromatogram of cellulose hydrolysis following the Saeman method , 10 g of Cellulose, acid concentration of 100 mM, temperature of 180ºC, black curve (Sample 2), rose curve (sample 3),blue curve (Sample 4)

62

The best result was achieved at the 7th sample 60 minutes from start with a concentration of 1.65 g/l of glucose representing a conversion of 8% of the cellulose into glucose, after that the glucose concentrations started to decrease slowly until the end of the experiment which can be seen by the 9th sample in the graphic.

As the method proved to be satisfactory with cellulose experiments were done with the 3 raw materials in study, and the best results of the whole trial were obtained. The results under the same conditions with 20 g of Marc 1 can be seen on graphic 19 and table 15.

Graphic 19. HPLC chromatogram of Marc 1 acid hydrolysis following the Saeman method , 20 g of Marc 1, acid concentration of 100 mM, temperature of 180ºC, black curve (Sample 2), rose curve (sample 4),blue curve (Sample 5)

63

Fermentable Time ( min ) Glucose (g/l) Xylose (g/l) Fructose (g/l) sugars (g) / g raw material

Sample 1 0 0 0 0 0 Sample 2 30 0.77 1.44 0.70 0.07 Sample 4 50 1.15 2.48 0.83 0.11 Sample 5 60 1.18 1.93 0.74 0.10 Sample 6 70 0.91 0.88 0.34 0.05 Sample 7 80 1.02 0 0 0.03 Sample 8 90 1.02 0 0 0.03 Sample 9 120 0 0 0 0

Table 15. Glucose, Xylose and Fructose concentrations and Fermentable sugar yield for each sample in the Marc 1 acid hydrolysis following the Saeman method

The concentration of xylose and fructose was maximized at 50 minutes from start corresponding to the 4th sample with the values of 2.48 g/l and 0.83 g/l respectively, the glucose values showed a maximum at the 5 th sample, with a value of 1.18 g/l nearly the same obtained at the 4 th sample, that overall corresponds to the sample where a higher a concentration of the 3 sugars was obtained. If the results are compared to the results obtained in the first trials using a much higher acid concentration, it suggests that the glucose obtained at this point of the hydrolysis corresponds to the easily hydrolysed cellulose, and most likely in order to obtain a higher glucose yield the reaction would have to continue for a longer period in order to hydrolyse the hard cellulose, which would end up in the complete loss of the xylose released from the hemicellulose. Also the sample times was completely changed, and one of the mistakes that was being done in the first trials was the completely wrong sampling time. Basically the xylose was being released and destroyed too quickly to be detected by the sampling pattern adopted at the first trials. In order to improve the overall sugar concentration , after the time correspondent to the 4 th sample, the solid phase would have to be separated and rehydrolised, transforming the process in a 2 step system according to what was previously explained.

The conditions adopted for marc 1 were used also with marc 2 (20 g) and tomato waste (20 g). The results for marc 2 can be seen on graphic 20 and table 16.

64

Graphic 20. HPLC chromatogram of Marc 2 acid hydrolysis following the Saeman method , 20g of Marc 2, acid concentration of 100 mM, temperature of 180ºC, black curve (Sample 2), rose curve (sample 4),blue curve (Sample 5)

Fermentable Time ( min ) Glucose (g/l) Xylose (g/l) Fructose (g/l) sugars (g)/ g raw material

Sample 1 30 0 2.80 0.62 0.09 Sample 2 40 0 3.43 0.78 0.11 Sample 3 50 0.75 3.54 0.90 0.13 Sample 4 60 0.98 1.69 0.82 0.09 Sample 5 70 0.73 1.04 0.48 0.06 Sample 6 80 0.91 0.78 0.40 0.05 Sample 7 90 0.84 0 0.31 0.03 Sample 8 100 0.91 0 0.31 0.03

Table 16. Glucose,Xylose and Fructose concentrations and Fermentable sugar yield for each sample taken in the Marc 2 acid hydrolysis following the Saeman method

65 The results obtained with marc 2 were similar to marc 1, although all maximum concentrations achieved were better. The concentration of xylose and fructose was maximized at sample 3 corresponding to a time of 50 minutes and the concentration of glucose was maximized at sample 4 with the time of 60 minutes from the beginning of the hydrolysis. It should be noted that the HPLC samples for this experiment were ran for around 50 minutes , in opposition to the 20 minutes of the samples of most of the previous experiments. The peaks at minute 29 and 40 correspond to HMF (hydroxymethylfurfural) and furfural respectively, the most important degradation products of glucose and xylose according to literature. The continuous increase in these peaks with time supports the theory that both xylose and glucose, as well as other sugars, are being released and immediately degradated into byproducts.

The final hydrolysis performed was with tomato waste ( under the same conditions as the 2 previous experiments) . The results are shown on the graphic 21 and table 17.

Graphic 21. HPLC chromatogram of tomato waste acid hydrolysis following the Saeman method , 20 g of tomato waste, acid concentration of 100 mM, temperature of 180ºC, black curve (Sample 3), rose curve (sample 4),blue curve (Sample 6), blue curve (Sample 7)

66

Fermentable Time ( min ) Glucose (g/l) Xylose (g/l) Fructose (g/l) sugars (g)/ g raw material

Sample 1 0 0 0 0 0 Sample 2 10 0 0 0 0 Sample 3 20 0.43 0.82 0.54 0.04 Sample 4 30 0.87 1.66 0.67 0.08 Sample 6 40 1.01 2.05 0.69 0.09 Sample 7 50 1.17 1.71 0.67 0.09 Sample 8 60 1.10 1.24 0.56 0.07 Sample 9 70 0.98 0.99 0.54 0.06

Table 17. Glucose, Xylose and Fructose concentrations and Fermentable sugar yield for each sample in the Tomato waste acid hydrolysis following the Saeman method

Once again the results were similar to the previous ones with the biggest concentration of glucose and xylose achieved at the 6 th sample corresponding to 40 minutes, and the glucose highest concentration achieved at the 7 th sample under the time of 50 minutes from the start. Once again the HMF and furfural peaks are present and continuously increasing over time.

67 5.3. Conclusions

In the end of the experiment, it can be concluded that the maximum sugar concentration achieved for Marc 1 was 4.46 g/l with a conversion of 11 % of the raw material (w/w) in fermentable sugars. For Marc 2 and tomato waste the maximum fermentable sugar concentration achieved was 5.19 g/l and 3.16 g/l respectively, representing a conversion of 13 and 9% respectively.

Although the results were not as expected, due to the available time and lack of knowledge on the process, as well as composition of the raw materials, it would be possible to initiate the second phase,the fermentation of these sugars with the propper strain in order to produce PHAs. Burkolderia sachari sp, according to literature is able to assimilate the 3 most important compounds produced. Although it should be taken into consideration that at least the furfural produced during the hydrolysis is highly toxic to the bacteria, and therefore some trials should be done in order to recognize if the concentrations achieved are too toxic for the growth with the desired strain.

Observing the results of the second hydrolysis it can be seen that the glucose concentration was rising until the end of the experiment. The experiment should have been performed for a longer span of time, in order to see the maximum glucose concentration achieved.

Different possibilities arise for a possible increase on the results. Following the method described in the theoretical part with the two step procedure it is probably possible to achieve much better results. In the last experiments under the Saeman method a high quantity of xylose was achieved using low acid concentrations and a reasonably high temperature. If after the point where the xylose was maximized the hydrolysis would be finished, the solid part removed and submitted to a new hydrolysis under a much higher acid concentration with the correct temperature, possibly the resistant cellulose would be broken into glucose. Most likely the cellullose obtained in the experiments with lower acid concentration is the readily hydrolysable part, that is released contemporarily with xylose. While in the first experiments with high acid concentration the cellulose is released after the xylose was released and degradated, being in the latter case the glucose concentration obtained much higher. The furfural and hemifurfural value should have been quantitatively analysed in order to quantify at least indirectly the degree of xylose and glucose degradation, and perform the needed calculations in order to determine the best parameters.

The high market value of furfural should be taken into account when considering the implementation of this process in a small scale industrial plant. After purification, this valuable byproduct could help to balance out the costs.

68 6. Final considerations

Polyhydroxyalkanoates, as well as some other bioplastics, are, nowadays seen as the future of the polymer industry. In the future it is expected that these plastics will completely replace the petrochemical plastics. The truly widespread applications of the biopolymers are still rare, but with the development of new production techniques as well as the lowering on the production cost, soon the bioplastics will conquer their place in our society. In a World increasingly concerned about environmental problems, bioplastics pose a durable, environmental friendly solution to some of the disposal problems we face nowadays. Much research still needs to be done, especially concerning the mcl-PHAs, if the objective is to enlarge the span of applications of this type of polymer. Every year new and important articles about polyhydroxyalkanoates developments and production techniques are published, proving that this is an area in constant growth. Hopefully soon PHAs will be in packages, in medical devices, used as water treatment products and even disposable clothes. When that happens our society will be on the way to a brighter, cleaner and self sustained future.

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