Optimization of bioplastics production from cheese whey

Dissertation presented in partial fulfillment of the requirements for the degree of Master in Biotechnology

Production of bacterial biopolymers from industrial fat-containing wastes

Dissertation presented in partial fulfilment of the Requirements for the Degree of Master in Biotechnology

Production and characterization of Chitin-Glucan Complex by Komagataella pastoris

This thesis was focused on the production, extraction and characterization of chitin:β-glucan complex (CGC). In this process, glycerol byproduct from the biodiesel industry was used as carbon source. The selected CGC producing yeast was Komagataella pastoris (formerly known as Pichia pastoris), due the fact that to achieved high cell densities using as carbon source glycerol from the biodiesel industry. Firstly, a screening of K. pastoris strains was performed in shake flask assays, in order to select the strain of K. pastoris with better performance, in terms of growth, using glycerol as a carbon source. K. pastoris strain DSM 70877 achieved higher final cell densities (92-97 g/l), using pure glycerol (99%, w/v) and in glycerol from the biodiesel industry (86%, w/v), respectively, compared to DSM 70382 strain (74-82 g/l). Based on these shake flask assays results, the wild type DSM 70877 strain was selected to proceed for cultivation in a 2 l bioreactor, using glycerol byproduct (40 g/l), as sole carbon source. Biomass production by K. pastoris was performed under controlled temperature and pH (30.0 ºC and 5.0, respectively). More than 100 g/l biomass was obtained in less than 48 h. The yield of biomass on a glycerol basis was 0.55 g/g during the batch phase and 0.63 g/g during the fed-batch phase. In order to optimize the downstream process, by increasing extraction and purification efficiency of CGC from K. pastoris biomass, several assays were performed. It was found that extraction with 5 M NaOH at 65 ºC, during 2 hours, associated to neutralization with HCl, followed by successive washing steps with deionised water until conductivity of ≤20μS/cm, increased CGC purity. The obtained copolymer, CGCpure, had a chitin:glucan molar ratio of 25:75 mol% close to commercial CGC samples extracted from A. niger mycelium, kiOsmetine from Kitozyme (30:70 mol%). CGCpure was characterized by solid-state Nuclear Magnetic Resonance (NMR) spectroscopy and Differential Scanning Calorimetry (DCS), revealing a CGC with higher purity than a CGC commercial (kiOsmetine). In order to optimize CGC production, a set of batch cultivation experiments was performed to evaluate the effect of pH (3.5–6.5) and temperature (20–40 ºC) on the specific cell growth rate, CGC production and polymer composition. Statistical tools (response surface methodology and central composite design) were used. The CGC content in the biomass and the volumetric productivity (rp) were not significantly affected within the tested pH and temperature ranges. In contrast, the effect of pH and temperature on the CGC molar ratio was more pronounced. The highest chitin: β-glucan molar ratio (> 14:86) was obtained for the mid-range pH (4.5-5.8) and temperatures (26–33 ºC). The ability of K. pastoris to synthesize CGC with different molar ratios as a function of pH and temperature is a feature that can be exploited to obtain tailored polymer compositions.(...)

Production of polyhydroxyalkanoates from oil-containing substrates

Different oil-containing substrates, namely, used cooking oil (UCO), fatty acids-byproduct from biodiesel production (FAB) and olive oil deodorizer distillate (OODD) were tested as inexpensive carbon sources for the production of polyhydroxyalkanoates (PHA) using twelve bacterial strains, in batch experiments. The OODD and FAB were exploited for the first time as alternative substrates for PHA production. Among the tested bacterial strains, Cupriavidus necator and Pseudomonas resinovorans exhibited the most promising results, producing poly-3-hydroxybutyrate, P(3HB), form UCO and OODD and mcl-PHA mainly composed of 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate (3HD) monomers from OODD, respectively. Afterwards, these bacterial strains were cultivated in bioreactor. C. necator were cultivated in bioreactor using UCO as carbon source. Different feeding strategies were tested for the bioreactor cultivation of C. necator, namely, batch, exponential feeding and DO-stat mode. The highest overall PHA productivity (12.6±0.78 g L-1 day-1) was obtained using DO-stat mode. Apparently, the different feeding regimes had no impact on polymer thermal properties. However, differences in polymer‟s molecular mass distribution were observed. C. necator was also tested in batch and fed-batch modes using a different type of oil-containing substrate, extracted from spent coffee grounds (SCG) by super critical carbon dioxide (sc-CO2). Under fed-batch mode (DO-stat), the overall PHA productivity were 4.7 g L-1 day-1 with a storage yield of 0.77 g g-1. Results showed that SCG can be a bioresource for production of PHA with interesting properties. Furthermore, P. resinovorans was cultivated using OODD as substrate in bioreactor under fed-batch mode (pulse feeding regime). The polymer was highly amorphous, as shown by its low crystallinity of 6±0.2%, with low melting and glass transition temperatures of 36±1.2 and -16±0.8 ºC, respectively. Due to its sticky behavior at room temperature, adhesiveness and mechanical properties were also studied. Its shear bond strength for wood (67±9.4 kPa) and glass (65±7.3 kPa) suggests it may be used for the development of biobased glues. Bioreactor operation and monitoring with oil-containing substrates is very challenging, since this substrate is water immiscible. Thus, near-infrared spectroscopy (NIR) was implemented for online monitoring of the C. necator cultivation with UCO, using a transflectance probe. Partial least squares (PLS) regression was applied to relate NIR spectra with biomass, UCO and PHA concentrations in the broth. The NIR predictions were compared with values obtained by offline reference methods. Prediction errors to these parameters were 1.18 g L-1, 2.37 g L-1 and 1.58 g L-1 for biomass, UCO and PHA, respectively, which indicates the suitability of the NIR spectroscopy method for online monitoring and as a method to assist bioreactor control. UCO and OODD are low cost substrates with potential to be used in PHA batch and fed-batch production. The use of NIR in this bioprocess also opened an opportunity for optimization and control of PHA production process.

Bioproduction of succinic acid from renewable feedstocks by Actinobacillus succinogenes 130Z

Succinic acid (SA) is a highly versatile building block that is used in a wide range of industrial applications. The biological production of succinic acid has emerged in the last years as an efficient alternative to the chemical production based on fossil fuels. However, in order to fully replace the competing petro-based chemical process from which it has been produced so far, some challenges remain to be surpassed. In particular, one main obstacle would be to reduce its production costs, mostly associated to the use of refined sugars. The present work is focused on the development of a sustainable and cost-e↵ective microbial production process based on cheap and renewable resources, such as agroindustrial wastes. Hence, glycerol and carob pods were identified as promising feedstocks and used as inexpensive carbon sources for the bioproduction of succinic acid by Actinobacillus succinogenes 130Z, one of the best naturally producing strains. Even though glycerol is a highly available carbon source, as by-product of biodiesel production, its consumption by A. succinogenes is impaired due to a redox imbalance during cell growth. However, the use of an external electron acceptor such as dimethylsulfoxide (DMSO) may improve glycerol metabolism and succinic acid production by this strain. As such, DMSO was tested as a co-substrate for glycerol consumption and concentrations of DMSO between 1 and 4% (v/v) greatly promoted glycerol consumption and SA production by this biocatalyst. Aiming at obtaining higher succinic acid yield and production rate, batch and fed-batch experiments were performed under controlled cultivation conditions. Batch experiments resulted in a succinic acid yield on glycerol of 0.95 g SA/g GLY and a production rate of 2.13 g/L.h, with residual production of acetic and formic acids. In fed-batch experiment, the SA production rate reached 2.31 g/L.h, the highest value reported in the literature for A. succinogenes using glycerol as carbon source. DMSO dramatically improved the conversion of glycerol by A. succinogenes and may be used as a co-substrate, opening new perspectives for the use of glycerol by this biocatalyst. Carob pods, highly available in Portugal as a residue from the locust bean gum industry, contain a significant amount of fermentable sugars such as sucrose, glucose and fructose and were also used as substrate for succinic acid production. Sugar extraction from raw and roasted carobs was optimized varying solid/water ratio and extraction time, maximizing sugar recovery while minimizing the extraction of polyphenols. Kinetic studies of glucose, fructose and sucrose consumption by A. succinogenes as individual carbon sources till 30 g/L were first determined to assess possible metabolic diferences. Results showed no significant diferences related to sugar consumption and SA production between the diferent sugars. Carob pods water extracts were then used as carbon source during controlled batch cultivations. (...)

Valorisation of side streams from wheat milling and confectionery industries for consolidated production and extraction of microbial lipids

Crude enzymes produced via solid state fermentation (SSF) using wheat milling by-products have been employed for both fermentation media production using flour-rich waste (FRW) streams and lysis of Rhodosporidium toruloides yeast cells. Filter sterilization of crude hydrolysates was more beneficial than heat sterilization regarding yeast growth and microbial oil production. The initial carbon to free amino nitrogen ratio of crude hydrolysates was optimized (80.2 g/g) in fed-batch cultures of R. toruloides leading to a total dry weight of 61.2 g/L with microbial oil content of 61.8 % (w/w). Employing a feeding strategy where the glucose concentration was maintained in the range of 12.2 – 17.6 g/L led to the highest productivity (0.32 g/L∙h). The crude enzymes produced by SSF were utilised for yeast cell treatment leading to simultaneous release of around 80% of total lipids in the broth and production of a hydrolysate suitable as yeast extract replacement.

Formulation of fermentation media from flour-rich waste streams for microbial lipid production by Lipomyces starkeyi

Flour-rich waste (FRW) and by-product streams generated by bakery, confectionery and wheat milling plants could be employed as the sole raw materials for generic fermentation media production, suitable for microbial oil synthesis. Wheat milling by-products were used in solid state fermentations (SSF) of Aspergillus awamori for the production of crude enzymes, mainly glucoamylase and protease. Enzyme-rich SSF solids were subsequently employed for hydrolysis of FRW streams into nutrient-rich fermentation media. Batch hydrolytic experiments using FRW concentrations up to 205 g/L resulted in higher than 90%(w/w) starch to glucose conversion yields and 40% (w/w) total Kjeldahl nitrogen to free amino nitro-gen conversion yields. Starch to glucose conversion yields of 98.2, 86.1 and 73.4% (w/w) were achieved when initial FRW concentrations of 235, 300 and 350 g/L were employed in fed-batch hydrolytic experiments, respectively. Crude hydrolysates were used as fermentation media in shake flask cultures with the oleaginous yeast Lipomyces starkeyi DSM 70296 reaching a total dry weight of 30.5 g/L with a microbial oil content of 40.4% (w/w), higher than that achieved in synthetic media. Fed-batch bioreactor cultures led to a total dry weight of 109.8 g/L with a microbial oil content of 57.8% (w/w) and productivity of 0.4 g/L/h.

Biorefinery development through utilization of biodiesel industry by-products as sole fermentation feedstock for 1,3-propanediol production

Rapeseed meal (RSM) hydrolysate was evaluated as substitute for commercial nutrient supplements in 1,3-propanediol (PDO) fermentation using the strain Clostridium butyricum VPI 1718. RSM was enzymatically converted into a generic fermentation feedstock, enriched in amino acids, peptides and various micro-nutrients, using crude enzyme consortia produced via solid state fermentation by a fungal strain of Aspergillus oryzae. Initial free amino nitrogen concentration influenced PDO production in batch cultures. RSM hydrolysates were compared with commercial nutrient supplements regarding PDO production in fed-batch cultures carried out in a bench-scale bioreactor. The utilization of RSM hydrolysates in repeated batch cultivation resulted in a PDO concentration of 65.5 g/L with an overall productivity of 1.15 g/L/h that was almost 2 times higher than the productivity achieved when yeast extract was used as nutrient supplement.

Rhodosporidium toruloides cultivated in NaCl-enriched glucose-based media: adaptation dynamics and lipid production

In the present report and for the first time in the international literature, the impact of the addition of NaCl upon growth and lipid production on the oleaginous yeast Rhodosporidium toruloides was studied. Moreover, equally for first time, lipid production by R. toruloides was performed under non-aseptic conditions. Therefore, the potentiality of R. toruloides DSM 4444 to produce lipid in media containing several initial concentrations of NaCl with glucose employed as carbon source was studied. Preliminary batch-flask trials with increasing amounts of NaCl revealed the tolerance of the strain against NaCl content up to 6.0% (w/v). However, 4.0% (w/v) of NaCl stimulated lipid accumulation for this strain, by enhancing lipid production up to 71.3% (w/w) per dry cell weight. The same amount of NaCl was employed in pasteurized batch-flask cultures in order to investigate the role of the salt as bacterial inhibiting agent. The combination of NaCl and high glucose concentrations was found to satisfactorily suppress bacterial contamination of R. toruloides cultures under these conditions. Batch-bioreactor trials of the yeast in the same media with high glucose content (up to 150 g/L) resulted in satisfactory substrate assimilation, with almost linear kinetic profile for lipid production, regardless of the initial glucose concentration imposed. Finally, fed-batch bioreactor cultures led to the production of 37.2 g/L of biomass, accompanied by 64.5% (w/w) of lipid yield. Lipid yield per unit of glucose consumed received the very satisfactory value of 0.21 g/g, a value amongst the highest ones in the literature. The yeast lipid produced contained mainly oleic acid and to lesser extent palmitic and stearic acids, thus constituting a perfect starting material for “second generation” biodiesel

Synthesis of biodegradable polyhydroxyalcanoate copolymer from a renewable source by alternate feeding

Polyhydroxyalcanoates copolymers with 3-hydroxybutirate (3HB) and 3-hydroxyvalerate (3HV) co-monomers, P3(HB-co-x%HV), were produced in fed-batch culture by Ralstonia eutropha DSM428 using fructose as a single carbon source in the first step and adding propionic acid in the second step by alternating feeding. Polymer yield was 0.18 g/L with a content of 24 mol% of the 3HV fraction determined by H-1 NMR. NMR measurements indicated that the polymer obtained is isotactic. The copolymer attained 35% of crystallinity according to X-ray diffraction measurements, and two (020) planes were observed. Thermal behavior presented melting temperature at 154 degrees C and the crystallization temperature was 65 degrees C. A glass transition temperature was observed at -10 degrees C. Average molecular weight measured by GPC was 4.9 x 10(5) Dalton. Isothermal radial growth rates of spherulites of P3(HB-co-24%HV) were studied. All experimental facts and the analysis of the sequence distribution of diads and triads of 3HB and 3HV units led to the conclusion that it is not a completely statistical random copolymer once it contains different types of segments. POLYM.

Biosynthesis and characterization of biodegradable Poly(3-hydroxybutyrate) from renewable sources

Poly(3-hydroxybutyrate) was produced in fed-batch cultures of Ralstonia eutropha DSM 428 and Alcaligenes latus ATCC 29712 on a mineral medium with different carbon sources such as sucrose, sodium lactate, lactic acid, soybean oil and fatty acid. The bacteria converted the different carbon sources supplied into P3HB. The best results were obtained when lactate or soybean oil were supplied as the sole carbon source. The range of number average molar mass (Mn) for the polymers, analyzed by Gel Permeation Chromatography was 1.65 to 0.79 x 10(5) g mol(-1). FTIR spectroscopy revealed a characteristic absorbance associated with polyester structures. The crystallinity degree, determinate from X-ray diffractograms, was about 69% in all synthesized polymers. The thermal properties associated to semicrystalline polymers indicated a glass transition at 0.1 degrees C and a melting point at about 175 degrees C and enthalpy of 63-89 J g(-1). The (1)H-NMR and (13)C-NMR spectra of the polymers were in agreement with the calculated chemical shifts associated with P3HB structures.

Estratégias de Produção in vitro de Bioinseticida Viral: influências do Isolado, da Cinética e do Modo de operação

Societal concerns about environmental sustainability has lead to the development of ecologically-friendly alternatives to chemical insecticides for crop protection. One such alternative is biological pest control. In particular, baculoviruses are well suited as insect biopesticides due to their narrow host specificity and relative ease of propagation. In Brazil, the baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) is the main biological control agent employed for the soybean pest, Anticarsia gemmatalis. This baculovirus biopesticide is currently produced using caterpillars, but increasing market demand for the product has encouraged the development of an in vitro manufacturing process, which can be scaled up to much higher virus productivities. In this study, three wild-type AgMNPV isolates (AgMNPV-2D, AgMNPV-MP2 and AgMNPV-MP5) and a recombinant form (vAgEGT-LacZ) were characterised in terms of occlusion body (OB) production and infection kinetics, to enable future optimisation of the in vitro production process. These viruses were propagated using a Spodoptera frugiperda (IPLB-SF21) insect cell line grown in shaker-flask batch cultures. Among the virus isolates tested, AgMNPV-MP5 was found to be the best producer, yielding (5.3±0.85)x108 OB/mL after 8 days post infection. The characterisation of vAgEGT-LacZ propagation in suspension cell cultures has not been previously reported in the literature; hence it became the main focus for this thesis. In particular, it was carried out a study on the effect of the multiplicity of infection (MOI) on OB production. Five successive batches were performed getting a final production (8.9±1.42)x1014 occlusion bodies, considering that production is related for a bioreactor with final volume of 10m3. A low MOI associated with a fed-batch process for vAgEGT-LacZ production was found to support a 3-fold higher OB yield when compared to the default batch process (1.8x107 and 5.3x107 OB/mL, respectively). This yield is competitive with regards to the production process.

Effect of C/N Ratio and Physicochemical Conditions on the production of Rhamnolipids by Pseudomonas Aeruginosa LBI

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

INFLUENCE of GLYCEROL and ORNITHINE FEEDING on CLAVULANIC ACID PRODUCTION BY Streptomyces clavuligerus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

CURRENT ASPECTS OF FUEL ETHANOL-PRODUCTION IN BRAZIL

Brazil has become a great producer of bioethanol using sugarcane as the basic raw material. Fed-batch process and continuous process are used. Biogas generation from vinasse, production of dry yeast, and autolyzed bagasse for animal feed are making the ethanol production less polluting and more profitable. Bagasse surplus has also been converted into electrical energy. Another alternative use for bioethanol is its conversion to petrochemical derivatives. Up to the present, however, this conversion has been carried out on only a small scale by the industry.