BIOMASS AND LIPID PRODUCTION FROM HETEROTROPHIC AND MIXOTROPHIC FED-BATCH CULTIVATIONS OF MICROALGAE \u3ci\u3eChlorella\u3c/i\u3e \u3ci\u3eprotothecoides\u3c/i\u3e USING GLYCEROL

Chlorella protothecoides is a microalga that can grow both photo-autotrophically and/or heterotrophically under different culture and environmental conditions. In this study both the heterotrophic growth and mixotrophic growth have been conducted. The heterotrophic experiments were conducted completely in the dark while the mixotrophic experiments had the dark cycles with periodic light exposure. The aim of the study was to independently understand the effect of each mode on biomass and lipid yields. For the heterotrophic experiments, glycerol was used as an external organic carbon source while yeast extract was used as the nitrogen source. The carbon and nitrogen source were added to a defined culture medium. Three different grades of glycerol were evaluated for their effect on the biomass and lipid yields in the heterotrophic experiments, with the 65% crude glycerol proving best giving an average biomass concentration of 22.13 ± 0.17 g/L and average lipid concentration of 9.75 ± 0.02 g/L at the end of an eight-day fed-batch fermentation. The average biomass concentrations did not increase after the eighth day of fermentation. The pH was maintained at a constant value of 6.8 and temperature at 280C. As the experiments were carried out in fed-batch mode, addition of the culture medium was done every 24 hours to maintain the carbon and nitrogen sources at 30g/L and 4g/L respectively till the eighth day. Yeast extract was found to be a good nitrogen source, as it also provides vitamins, amino acids and important growth factors as oppose to some other sources like ammonia and urea (Shi et al., 2000; Gonzalez-Bashan et al., 2000; Illman et al., 2000; Chen et al., 2006). The mixotrophic experiments were aimed to expose the algae to alternating light and dark cycles to enhance biomass accumulation during light cycle and lipid accumulation during dark cycles. The light cycle help to assimilate CO2 and produce energy via photosynthesis, which comprises the catabolic reaction, while the switch to the dark cycle allows anabolic reactions where accumulation of lipids and production of other compounds occur. Here, the algae were exposed to light for 8 hours and dark for 16 hours each day for eight days. The 65% crude glycerol was supplemented as the external carbon source to be utilized by the algae during the dark cycles while yeast extract was used as the nitrogen source. Here the average maximum biomass concentration of 28.95 ± 0.26 g/L and the average lipid concentration of 13.14 ± 0.01 g/L were obtained which were found to be higher than the heterotrophic results. With intermittent light exposure, the lipid yields were found to increase from a maximum of 0.44 ± 0.004 gram lipid/gram biomass for heterotrophic experiments to 0.46 ± 0.004 gram lipid/gram biomass for mixotrophic experiments. The mixotrophic experiments also provided an increase in the average maximum overall biomass concentration from 22.13 ± 0.17 g/L in heterotrophic to 28.95 ± 0.26 g/L in mixotrophic experiments

Production and characterization of value-added biorenewable chemicals

Nisin is the only FDA-approved bacteriocin with a GRAS (Generally Regarded as Safe) status, used as a biopreservative/shelf-life extender in foods and many other products including pharmaceutical, veterinary and health care products. It is produced commercially by fermentation of a milk-based medium, using strains of Lactococcus lactis subsp. lactis. However, being a dairy-based product requires its labeling since milk is an allergen. Also, since its high production cost is mainly dominated by its medium costs, an emerging trend has been observed with a growing number of studies aimed at finding alternative, non-dairy and preferably non-allergen-based substrates. In order to cut costs, and to also be an environmentally friendly approach, several of these substrates have been sourced from agriculture or aquaculture-based waste streams. The review included in this dissertation highlights the advantages of value-addition to waste and the promising potentials of some of these low-value nutritive sources for the production of high-value nisin. An in-depth study of soy whey (SW) as a non-dairy, nisin fermentation feedstock was performed using Lactococcus lactis. Results indicated that SW was able to produce equivalent biomass and nisin yields (2.18g/L and 619mg/L), as compared to the commercial medium, de Man-Rogosa-Sharpe (MRS) broth (2.17g/L and 672mg/L, respectively), without the need for external nutrient supplementation. This indicated the nutritive qualities of this co-product stream in being able to support the growth of a fastidious bacterial culture. The success of SW as a bacterial growth medium motivated the second study which focused on growing the oleaginous algal strain Chlorella vulgaris under mixotrophic and heterotrophic culture conditions. Microalgae are cultivated in large scale for several commercially important products among which algal lipids have gained increasing interest due to their use as biodiesel feedstock as well as a source of essential fatty acids with neutraceutical value. Photoautotrophic algal cultivation suffers from low growth rates while heterotrophic/mixotrophic modes have high media costs. In order to boost algal lipid production with lower media costs, two co-product streams, SW and thin stillage (TS) were tested as growth substrates. TS is a high-organic strength co-product generated from the dry-grind corn-ethanol industry. Traditional use of TS involves an energy-intensive concentration process to form syrup which finally ends up as animal feed in Distillers\u27 Dried Grains with Solubles (DDGS). The cost-effectiveness and success of the corn-ethanol industry is highly dependent on the value of its co-products. Therefore, in order to be an economically viable and an environmentally sustainable process, researching new avenues for value-addition to TS is very important. As per the results from the current study, biomass yields (dry basis, db) from TS, SW and a synthetic control medium-MBM (modified basal medium) after 4 days of incubation were 9.8, 6.3 and 8.0 g/L with oil contents at 43, 11, and 27 % (w/w) respectively. Polyunsaturated fatty acids (PUFAs) or essential FAs, were found to be highest in Chlorella-TSoil (56%), followed by 38% in Chlorella-SWoil and 31% in Chlorella-MBMoil. Therefore, mixotrophic cultivation of C. vulgaris in TS and SW produced high yields of both algal biomass and lipids at low cost, thus adding value to co-product streams and improving economic viability of algal cultivation. The high algal oil yields from TS motivated the third study involving the use of oleaginous fungal strain, Mucor circinelloides for its potential in adsorbing/assimilating the oil and nutrients present in TS, for the production of lipid and protein-rich fungal biomass. Fungal batch cultivation for 2 days using a 6-L airlift reactor led to a 92% increase in oil yield from TS, relative to the original oil content, with concomitant reduction in suspended solids and chemical oxygen demand (COD) in TS by 95% and 89% respectively. M. circinelloides, when grown on TS gave a biomass yield of 20 g/L (dry basis), with a lipid content of 46 % (dwb). The polyunsaturated fatty acids were 52% of the total lipids. Overall, fungal cultivation on TS produced a high-protein animal feed and high-value fungal oil, thus improving corn-ethanol process economics. The fourth study focused on employing the hurdle concept and developing more efficient antimicrobial systems by combining plant essential oils (EOs) with food-grade organic acids. The seven most active oils among the sixteen tested, were combined with five different organic acids and the antimicrobial interactions (synergistic/additive/antagonistic) were examined against food-related pathogens. Malic and citric acid were the most inhibitory acids which showed synergism with mountain savory, redistilled oregano (RO), and cinnamon oils against S.aureus 25923 and with cassia, RO, and cinnamon oils against E.coli 25922. The only synergistic EO-acid combination against C.albicans 10231 was lemon myrtle-citric acid

Anaerobic processes for waste biomass treatment: applications and mathematical modeling

The increasing global energy demand and depletion of fossil fuels are driving international policies to promote the use of alternative energy sources, leading to the mitigation of global warming and greenhouse gas (GHG) emissions. In this context, the sustainable valorisation via biological anaerobic processes of organic waste biomasses represents a promising technology for producing renewable energy and value added chemicals in a biorefinery concept. Anaerobic Digestion (AD) and Dark Fermentation (DF) are the most explored biological routes to produce renewable energy in the form of methane and hydrogen gas, respectively. Due to its high flexibility and applicability to a wide range of organic substrates, AD has been largely adopted in full scale applications, with increasing interest from both academia and industries. Conversely, low biohydrogen yields and incomplete biomass conversion limit DF scaled-up applications as a self-standing biotechnology for energy production. Moreover, the combination of different anaerobic processes, such as DF-AD or DF-Photo-Fermentation (PF), might be adopted to overcome the main drawbacks deriving from the application of DF processes. A significant example is represented by the use of DF by-products, such as organic acids and alcohols, for H2 production via PF. The combined DF-PF process results in the increase of the total hydrogen yield (usually evaluated in terms of mol H2 mol C6H12O6-1) and the production of poly-β-hydroxybutyrate (PHB), which are stored by Purple Non-Sulphur Bacteria, performing the PF step, as a carbon reserve available under nutrient starvation. In this thesis, different anaerobic technologies have been applied to achieve higher biogas yields and/or by-products valorisation in a biorefinery concept. In particular, the enhancement of AD performances, in terms of biogas productivity, has been addressed by optimizing the crucial start-up phase of an anaerobic fluidized bed reactor. The stability of DF and enhancement of biohydrogen yields have been obtained by optimizing the operative conditions of a DF reactor continuously fed with cheese whey in thermophilic regimen, and by adopting a combined DF-PF configuration for the energetic valorisation of the organic fraction of municipal solid wastes, respectively. Contextually, the valorisation of Dark Fermentation effluents (DFE) was achieved by i) producing PHB via PF, ii) extracting value added acids with anionic resins or iii) using DFE as a complex media for the dissolution of recalcitrant materials. Furthermore, a 1-D mathematical model has been presented to analyse and predict the microbial colonization of anaerobic multispecies biofilms

Analysis and modelling of microalgae growth and production of high added-value metabolites

Microalgae are a very versatile microorganism that have the ability to modify their biomass composition under controlled condition in order to accumulate products having applications in several sectors. The aim of this thesis work is the analysis and modelling of both microalgal growth and production of high added-value metabolites, focusing also on their extraction and purification. An outdoor 10 bubble column photobioreactors (PBRs) pilot plant for the cultivation of two microalgae named Tetradesmus obliquus and Graesiella emersonii, covering a 9 months cultivation period (March 2017-December 2017), has been installed and operated. All collected data (as microalgal growth rate, outdoor parameters and initial cultivation’s conditions) have been used to develop an empirical model for prediction of microalgal growth in photobioreactors at specific outdoor conditions, using Principal Component Analysis and Partial Least Squares regression method, obtaining acceptable outcomes for both responses: microalgal specific growth rate (μ) and maximum productivity (Pmax). Concerning microalgal metabolism, also a new mathematical model able to represent in a simple way the accumulation of metabolites inside microalgae, focusing on the carbon partitioning process between triacylglycerides (TAG) and starch during nitrogen starvation in phototrophy, has been developed, obtaining high R-Squared values as index of model’s goodness of fitting. A future application of these models can be found in the MEWLIFE European project, in which Bio-P has a role as partner, since this project has as aim the production of microalgal biomass in an integrated phototrophic and heterotrophic cultivation system using preconcentrated olive oil wastewaters (OMWW) as carbon source. As a completion of the microalgal process treatment, a study of the downstream processes for the extraction (using supercritical CO2) and purification of the high added value metabolites (with molecular distillation) has been carried out, developing a feasibility study also from the economical point of view. As regard the supercritical CO2 the best extraction conditions in terms of operative variables have been: T = 60°C, P = 250 bar and SSR = 5 h−1 with a daily amount of the desired products equal to 147 kg and OPEX = 561.7 k€/year and CAPEX = 2717.9 k€/year. Regarding the molecultar distillation process, the best operating conditions have been found at T = 128 °C and P = 0.33 Pa, obtaining OPEX = 498.23 k€/year and CAPEX = 2387.4 k€/year

Development of universal stoichiometric coefficients for modeling microalgal cultivation systems

Biological treatment processes at water resource recovery facilities (WRRFs; a.k.a. wastewater treatment plants) are approaching the limit of technology for nitrogen and phosphorus removal. Algae treatment technologies have the ability to remove additional nitrogen and phosphorus, thereby lowering the effluent nutrient discharge level at WRRFs. A critical challenge for the adoption of algal technologies, however, is the lack of robust algae modeling platforms for wastewater treatment that can predict process performance under fluctuating reactor conditions and despite the inevitable biodiversity of influent wastewater. One necessary step towards improved modeling capabilities for algae treatment systems is the development of generalizable model parameters, such as stoichiometric parameters – like those used in the International Water Association’s (IWA’s) Activated Sludge Models (ASMs). This work introduces universal stoichiometric coefficients for algal process modeling derived from the conserved enzymatic properties for seven algae species using 11 genome-scale models. The model parameters include yield coefficients for algae grown under various energy inputs (photoautotrophic and heterotrophic), nitrogen sources (ammonia and nitrate), and carbon sources (inorganic, acetate, and glucose) as well as stoichiometric parameters for the accumulation of storage compounds (starch and lipids). Generalizable stoichiometric parameters based on conserved metabolic properties would bolster accuracy and the accessibility of algal process models. This will help promote the use of algal technologies by wastewater design engineers and utilities to improve the effluent quality at water resource recovery facilities

Factors Affecting Microalgae Production for Biofuels and the Potentials of Chemometric Methods in Assessing and Optimizing Productivity

Microalgae are swift replicating photosynthetic microorganisms with several applications for food, chemicals, medicine and fuel. Microalgae have been identified to be suitable for biofuels production, due to their high lipid contents. Microalgae-based biofuels have the potential to meet the increasing energy demands and reduce greenhouse gas (GHG) emissions. However, the present state of technology does not economically support sustainable large-scale production. The biofuel production process comprises the upstream and downstream processing phases, with several uncertainties involved. This review examines the various production and processing stages, and considers the use of chemometric methods in identifying and understanding relationships from measured study parameters via statistical methods, across microalgae production stages. This approach enables collection of relevant information for system performance assessment. The principal benefit of such analysis is the identification of the key contributing factors, useful for decision makers to improve system design, operation and process economics. Chemometrics proffers options for time saving in data analysis, as well as efficient process optimization, which could be relevant for the continuous growth of the microalgae industry

Systems Metabolic Engineering of Microbial Cell Factories for the Synthesis of Value-added Chemicals

Microbial cell factories offer us an excellent opportunity for the conversion of many different cheaply available raw materials into valuable chemicals. Systems metabolic engineering aims at developing rational strategies for the engineering of microbial hosts by providing global level information of a cell. This dissertation focuses on metabolic engineering, bioprocess modeling and pathway analysis, to develop robust microbial cell factories for the synthesis of value-added chemicals. The following research tasks were completed in this regard. First, statistical models were developed for the prediction of product yields in engineered microbial cell factories - Saccharomyces cerevisiae and Escherichia coli: Chapter 2). A large space of experimental data for chemical production from recent references was collected and a statistics-based model was developed to calculate production yield. The input variables: numerical or categorical variables) for the model represented the number of enzymatic steps in the biosynthetic pathway of interest, metabolic modifications, cultivation modes, nutrition and oxygen availability. In addition, the use of 13C-isotopomer analysis method was proposed for the accurate determination of product yields in engineered microbes under complex cultivation conditions: Chapter 3). Second, metabolic engineering of the cyanobacterium, Synechocystis sp. PCC 6803 was performed for synthesizing isobutanol under phototrophic conditions: Chapter 4). With the expression of the heterologous genes from the Ehrlich Pathway, by incorporating an in situ isobutanol harvesting system, and also by employing mixotrophic conditions, the engineered Synechocystis 6803 strain accumulated a maximum of ~300 mg/L of isobutanol in a 21 day culture. In addition, Synechocystis 6803 was engineered for the synthesis of D-lactic acid: Chapter 5), via overexpression of a novel D-lactate dehydrogenase: encoded by gldA101). The production of D-lactate was further improved by employing three strategies:: i) cofactor balancing,: ii) codon optimization, and: iii) process optimization. The engineered Synechocystis 6803 produced 2.2 g/L D-lactate under photoautotrophic conditions with acetate, the highest reported lactate titer among all known cyanobacterial strains. Finally, an E. coli cell factory was engineered to study the fermentation kinetics for scaled-up isobutanol production: Chapter 6). Through kinetic modeling: to describe the dynamics of biomass, products and glucose concentration) and isotopomer analysis, we have also offered metabolic insights into the performance trade-off between two engineered isobutanol producing E. coli strains: a high performance and a low performance strain). The kinetic model can also predict isobutanol production under different fermentation conditions. I and my colleagues have also demonstrated that E. coli cell factory can also be used for converting waste acetate into free fatty acids through metabolic engineering. In conclusion, the opportunities and commercial limitations with current biotechnology as well as the role of systems metabolic engineering for the development of high performance microbial cell factories were discussed: Chapter 7)

Investigation of the growth, motility, and optical properties of microorganisms in active fluids

To have better control over photobioreactors at various operating conditions, it is necessary to characterize microorganisms’ motion, optimize light distribution, and investigate efficient mixing methods in photobioreactors. The second chapter of this thesis aims to develop a theoretical model for the calculation of microorganisms’ optical characteristics. Modeling light transfer in photobioreactors needs accurate input data to solve Maxwell’s equations. Here, input data include absorption properties of the microorganism’s pigment, pigment-content measurement, and the details of the shape and size of the microorganism cells. These input data predicted the optical characteristics of microorganism cells with homogeneous, coated, and heterogeneous geometries. The third chapter reports on experiments that were carried out to investigate the effect of two mixing methods, turbulent stirring and orbitally shaking, on the growth metrics of Synechocystis sp. CPCC 534, and compare them with stationary cultures. The study revealed that stirring Synechocystis cultures can enhance the growth rate, doubling per day, yield, and Chla production in contrast to cultures without any mixing. In the fourth chapter, the motility of wild-type Synechocystis sp. CPCC 534 was investigated to establish a correlation between the evolution of cell motility and cell growth phases during the complete growth cycle of 78 days. Average cell velocity, mean squared displacement (MSD), diffusion coefficient, and displacement probability density function (PDF) were calculated to assess the dynamics of Synechocystis sp. CPCC 534 during the growth period. The obtained results indicate that the age of microorganisms has a notable influence on different aspects of cell motility. Consequently, this can affect the transport characteristics of active suspension. In the final chapter of this thesis, we aimed to examine the transport characteristics of active fluids and passive fluids in a bifurcated microchannel with a rectangular cross-section. A PDMS microchannel was designed and fabricated to investigate the behavior of two fluids in the bifurcated microchannel. Finally, our investigation revealed that passive fluids exhibit higher velocity than active fluids. This difference arises due to the minimal movement of active fluids caused by their run-and-tumble motion

Isolation and identification of native microalgae for biodiesel production

La demande croissante en carburants, ainsi que les changements climatiques dus au réchauffement planétaire poussent le monde entier à chercher des sources d’énergie capables de produire des combustibles alternatifs aux combustibles fossiles. Durant les dernières années, plusieurs sources potentielles ont été identifiées, les premières à être considérées sont les plantes oléagineuses comme source de biocarburant, cependant l’utilisation de végétaux ou d’huiles végétales ayant un lien avec l’alimentation humaine peut engendrer une hausse des prix des denrées alimentaires, sans oublier les questions éthiques qui s’imposent. De plus, l'usage des huiles non comestibles comme sources de biocarburants, comme l’huile de jatropha, de graines de tabac ou de jojoba, révèle un problème de manque de terre arable ce qui oblige à réduire les terres cultivables de l'industrie agricole et alimentaire au profit des cultures non comestibles. Dans ce contexte, l'utilisation de microorganismes aquatiques, tels que les microalgues comme substrats pour la production de biocarburant semble être une meilleure solution. Les microalgues sont faciles à cultiver et peuvent croitre avec peu ou pas d'entretien. Elles peuvent ainsi se développer dans des eaux douces, saumâtres ou salées de même que dans les terres non cultivables. Le rendement en lipide peut être largement supérieur aux autres sources de biocarburant potentiel, sans oublier qu’elles ne sont pas comestibles et sans aucun impact sur l'industrie alimentaire. De plus, la culture intensive de microalgues pour la production de biodiesel pourrait également jouer un rôle important dans l'atténuation des émissions de CO2. Dans le cache de ce travail, nous avons isolé et identifié morphologiquement des espèces de microalgues natives du Québec, pour ensuite examiner et mesurer leur potentiel de production de lipides (biodiesel). L’échantillonnage fut réalisé dans trois régions différentes du Québec: la région de Montréal, la gaspésie et le nord du Québec, et dans des eaux douces, saumâtres ou salées. Cent souches ont été isolées à partir de la région de Montréal, caractérisées et sélectionnées selon la teneur en lipides et leur élimination des nutriments dans les eaux usées à des températures différentes (10 ± 2°C et 22 ± 2°C). Les espèces ayant une production potentiellement élevée en lipides ont été sélectionnées. L’utilisation des eaux usées, comme milieu de culture, diminue le coût de production du biocarburant et sert en même temps d'outil pour le traitement des eaux usées. Nous avons comparé la biomasse et le rendement en lipides des souches cultivées dans une eau usée par apport à ceux dans un milieu synthétique, pour finalement identifié un certain nombre d'isolats ayant montré une bonne croissance à 10°C, voir une teneur élevée en lipides (allant de 20% à 45% du poids sec) ou une grande capacité d'élimination de nutriment (>97% d'élimination). De plus, nous avons caractérisé l'une des souches intéressantes ayant montré une production en lipides et une biomasse élevée, soit la microalgue Chlorella sp. PCH90. Isolée au Québec, sa phylogénie moléculaire a été établie et les études sur la production de lipides en fonction de la concentration initiale de nitrate, phosphate et chlorure de sodium ont été réalisées en utilisant de la méthodologie des surfaces de réponse. Dans les conditions appropriées, cette microalgue pourrait produire jusqu'à 36% de lipides et croitre à la fois dans un milieu synthétique et un milieu issu d'un flux secondaire de traitement des eaux usées, et cela à 22°C ou 10°C. Ainsi, on peut conclure que cette souche est prometteuse pour poursuivre le développement en tant que productrice potentielle de biocarburants dans des conditions climatiques locales.The continuing increase in fuel demands, the dramatic situation in climate changes and the global warming are bringing the worldwide attention to the identification of alternative energy source for the production of combustibles that can replace fossil fuel. In last years, a lot of potential sources have been identified: the first potential biofuel feedstock that have been evaluated were oleaginous plants, but the utilization of vegetable, or vegetable oils, that may also be used for human feeding, could lead to the increase of food-grade oils costs and also generate ethic questions. Nevertheless, also using as biofuel sources not-edible oils, like oils from jatropha, tobacco seed or jojoba, the common problem for both edible and not-edible crops is the need to subtract arable land from agriculture and food industry. In this context, the utilization of aquatic microorganisms like microalgae as substrate for the production of biofuel seems to be the better solution. Microalgae are easy to cultivate and can grow with little or no attention, they can grow in fresh, brackish or salt water and in non-arable lands, moreover they are not edible with no consequences on food industry, and the oil productivity, with respect to the other potential biofuel sources, can be much higher. In addition, the intensive cultivation of microalgae for biodiesel production could also play an important role in CO2 mitigation. In this study, we isolated and morphologically identified Québec native micro algal species, surveyed and screened their potential for lipid (biodiesel) production. The sampling efforts made in three different regions of Québec: Montreal area, Gaspesie and Northern of Quebec; on fresh, brackish or saline water. One hundred strains were isolated from the Montreal area, characterized and screened for their lipid content and wastewater nutrient removal under different temperatures (10±2 ºC and 22±2 ºC). The high potential lipid producing algal species were selected. The use of wastewater as a substrate media decreases the economic cost realted to the biofuel production from microalgae as well as an interesting tool for wastewater treatment. We compared the biomass and lipid productivity of these strains on wastewater to a synthetic medium and identified a number of isolates that showed good growth at 10 ºC, gave a high lipid content (ranging from 20% to 45% of dry weight) or a high capacity for nutrient removal (>97% removal). Furthermore, we characterized one of the interesting strains that revealed high lipid and biomass productivity, the novel microalga Chlorella sp. PCH90. Its molecular phylogeny was established and lipid production studies as a function of the initial concentrations of nitrate, phosphate, and sodium chloride were carried out using Response Surface Methodology. Under the appropriate conditions this microalga could produce up to 36% lipid and grew well in both synthetic medium and secondary effluent from a wastewater treatment plant at both 22°C and 10°C. Thus, this strain is promising for further development as a potential biofuels producer under local climatic conditions

Systematic analysis of anaerobic conversion of microalgal biomass into biomethane aiming for process efficiency optimization

Klassen V. Systematic analysis of anaerobic conversion of microalgal biomass into biomethane aiming for process efficiency optimization. Bielefeld: Universität Bielefeld; 2017.Worldwide depletion of fossil fuel reserves advanced the search for environmental friendly and sustainable alternatives. The fact that microalgae perform very efficiently photosynthetic conversion of sunlight into chemical energy has moved them into the focus of regenerative fuel research, especially since algae cultivation, in contrast to land plants, is not restricted to arable land. Renewable fuel generation via anaerobic fermentation using microalgae biomass for biogas production, compared to biodiesel and bioethanol, is less intensive investigated. This thesis provides a systematic analysis of parameters influencing the degradability of microalgae biomass in an anaerobic digestion process, with respect to algae species, biomass composition and culture conditions. The biodegradability of twenty different freshwater microalgae species possessing different cell wall characteristics, cultured under comparable conditions and harvested in the same growth phase, was observed to be relatively similar, corresponding to rather low conversion efficiencies of less than 53 % of the theoretical maximum. These findings suggested that the recalcitrance of the cell wall is not the only factor influencing anaerobic digestion, since not every algal species contains a rigid cell wall, further indicating that other parameters must influence the accessibility of algae cells towards decomposition by anaerobic microorganisms. Naturally occurring nutrient starvation is a direct consequence of algae blooms in late summer, and therefore this natural phenomenon was simulated under controlled conditions and the impact on algae biomass degradability was investigated. Three scientifically and industrially relevant algae strains Chlamydomonas reinhardtii, Parachlorella kessleri and Scenedesmus obliquus were therefore cultured in low-nitrogen media (containing insufficient nitrogen source for extensive cell proliferation) and subjected at different growth stages to anaerobic fermentation in batch test. The results revealed a strong correlation of the cell starvation status and biodegradability to biogas, towards complete biomass disintegration at the maximum starvations level (indicated by max. C:N ratio). The feasibility of fermentation of “nitrogen starved” vs “nitrogen replete” microalgae biomass was furthermore investigated in a long term (160 days) continuous lab-scale simulation of an industrial biogas plant. The results of “nitrogen replete” biomass fermentation revealed low conversion efficiency and subsequent fermentations failure caused by high protein content in the biomass. The fermentation of “low nitrogen” biomass, on the contrary, was characterized by very stable process parameters and highly efficient biomass to methane conversion efficiency of 84 %. In comparison to “energy crops” (e.g. maize), usually used for biogas generation, the achieved methane yield was 37 % higher on biomass basis and approximately 4.5 times higher based on areal productivity (conservative estimation). In conclusion, this PhD work provides a simple and effective microalgae cultivation method for subsequent use of biomass as mono-substrate for anaerobic fermentation to methane. Highly efficient and stable fermentation process of this biomass was demonstrated in a continuous long-term experiment within this work and enables therefore an efficient industrial scale application