Continuous microalgae cultivation for wastewater treatment - Development of a process strategy during day and night.

Conventional wastewater treatment (WWT) is not able to recycle nutrients from the wastewater (WW) directly. Microalgae integrate the valuable nutrients nitrogen and phosphorus within their biomass very efficiently, making them predestined for an application in WWT. Nevertheless, microalgae-based processes are driven by natural sunlight as energy source, making a continuous process mode during day and night difficult. The aim of this study was therefore to investigate metabolic activities of the continuously cultivated microalgae Chlorella vulgaris at light and dark periods (16 h,8 h) with focus on nutrient uptake during night from a synthetic WW. Varying the dilution rate D (D = 0.0-1.0 d-1 in 0.1 d-1-steps) causes different limitations for algae growth. Nutrient limitations at low D's cause maximum accumulation of intracellular storage components (sum of carbohydrates and lipids) of ~70 % of dry biomass, starch is converted to lipids at the absence of light. From middle to high D's, the growth rate is determined by light limitation, reducing the intracellular storage components to ~20 % of dry biomass. Complete nutrient uptake is measurable up to D = 0.5 d-1, marking the maximum operating point for wastewater purification. At that point, cells are characterised by high protein (up to 57%DBM) and pigment (up to 6.9%DBM) quotas. During the night, the build-up of proteins at the degradation of intracellular storage components is furthermore visible. Applying the concept of active biomass (cells without storage components), a constant cellular protein (~68%ABM) and nitrogen quota (11.94%ABM) was revealed. A nitrogen spiking experiment clearly showed nitrogen uptake and proliferation during the night period. Based on the experimental data, a window of operation for a continuous WWT process was designed, allowing the hypothesis that continuous WWT using microalgae during day and night operation is possible without the supplementation of artificial light. This revealed the system's capacity to treat WW throughout 24 h applying cell recycling and storage of carbohydrate-rich biomass. At the end of the night, protein-rich biomass is available for further valorisation.

Characterization of an aerated submerged hollow fiber ultrafiltration device for efficient microalgae harvesting.

The present work characterizes a submerged aerated hollow fiber polyvinylidene fluorid (PVDF) membrane (0.03 µm) device (Harvester) designed for the ultrafiltration (UF) of microalgae suspensions. Commercial baker's yeast served as model suspension to investigate the influence of the aeration rate of the hollow fibers on the critical flux (CF, J c) for different cell concentrations. An optimal aeration rate of 1.25 vvm was determined. Moreover, the CF was evaluated using two different Chlorella cultures (axenic and non-axenic) of various biomass densities (0.8-17.5 g DW/L). Comparably high CFs of 15.57 and 10.08 L/m/2/h were measured for microalgae concentrations of 4.8 and 10.0 g DW/L, respectively, applying very strict CF criteria. Furthermore, the J c-values correlated (negative) linearly with the biomass concentration (0.8-10.0 g DW/L). Concentration factors between 2.8 and 12.4 and volumetric reduction factors varying from 3.5 to 11.5 could be achieved in short-term filtration, whereat a stable filtration handling biomass concentrations up to 40.0 g DW/L was feasible. Measures for fouling control (aeration of membrane fibers, periodic backflushing) have thus been proven to be successful. Estimations on energy consumption revealed very low energy demand of 17.97 kJ/m3 treated microalgae feed suspension (4.99 × 10-3 kWh/m3) and 37.83 kJ/kg treated biomass (1.05 × 10-2 kWh/kg), respectively, for an up-concentration from 2 to 40 g DW/L of a microalgae suspension.

Evaluation of Downstream Processing, Extraction, and Quantification Strategies for Single Cell Oil Produced by the Oleaginous Yeasts Saitozyma podzolica DSM 27192 and Apiotrichum porosum DSM 27194.

Single cell oil (SCO) produced by oleaginous yeasts is considered as a sustainable source for biodiesel and oleochemicals since its production does not compete with food or feed and high yields can be obtained from a wide variety of carbon sources, e.g., acetate or lignocellulose. Downstream processing is still costly preventing the broader application of SCO. Direct transesterification of freeze-dried biomass is widely used for analytical purposes and for biodiesel production but it is energy intensive and, therefore, expensive. Additionally, only fatty acid esters are produced limiting the subsequent applications. The harsh conditions applied during direct esterification might also damage high-value polyunsaturated fatty acids. Unfortunately, universal downstream strategies effective for all yeast species do not exist and methods have to be developed for each yeast species due to differences in cell wall composition. Therefore, the aim of this study was to evaluate three industrially relevant cell disruption methods combined with three extraction systems for the SCO extraction of two novel, unconventional oleaginous yeasts, Saitozyma podzolica DSM 27192 and Apiotrichum porosum DSM 27194, based on cell disruption efficiency, lipid yield, and oil quality. Bead milling (BM) and high pressure homogenization (HPH) were effective cell disruption methods in contrast to sonification. By combining HPH (95% cell disruption efficiency) with ethanol-hexane-extraction 46.9 ± 4.4% lipid/CDW of S. podzolica were obtained which was 2.7 times higher than with the least suitable combination (ultrasound + Folch). A. porosum was less affected by cell disruption attempts. Here, the highest disruption efficiency was 74% after BM and the most efficient lipid recovery method was direct acidic transesterification (27.2 ± 0.5% fatty acid methyl esters/CDW) after freeze drying. The study clearly indicates cell disruption is the decisive step for SCO extraction. At disruption efficiencies of >90%, lipids can be extracted at high yields, whereas at lower cell disruption efficiencies, considerable amounts of lipids will not be accessible for extraction regardless of the solvents used. Furthermore, it was shown that hexane-ethanol which is commonly used for extraction of algal lipids is also highly efficient for yeasts.

Microalgal kinetics - a guideline for photobioreactor design and process development.

Kinetics generally describes bio-(chemical) reaction rates in dependence on substrate concentrations. Kinetics for microalgae is often adapted from heterotrophs and lacks mechanistic foundation, e.g. for light harvesting. Using and understanding kinetic equations as the representation of intracellular mechanisms is essential for reasonable comparisons and simulations of growth behavior. Summarizing growth kinetics in one equation does not yield reliable models. Piecewise linear or rational functions may mimic photosynthesis irradiance response curves, but fail to represent the mechanisms. Our modeling approach for photoautotrophic growth comprises physical and kinetic modules with mechanistic foundation extracted from the literature. Splitting the light submodel into the modules for light distribution, light absorption, and photosynthetic sugar production with independent parameters allows the transfer of kinetics between different reactor designs. The consecutive anabolism depends among others on nutrient concentrations. The nutrient uptake kinetics largely impacts carbon partitioning in the reviewed stoichiometry range of cellular constituents. Consecutive metabolic steps mask each other and demand a maximum value understandable as the minimum principle of growth. These fundamental modules need to be clearly distinguished, but may be modified or extended based on process conditions and progress in research. First, discussion of kinetics helps to understand the physiological situation, for which ranges of parameter values are given. Second, kinetics should be used for photobioreactor design, but also for gassing and nutrient optimization. Numerous examples are given for both aspects. Finally, measuring kinetics more comprehensively and precisely will help in improved process development.

Mono- and dichromatic LED illumination leads to enhanced growth and energy conversion for high-efficiency cultivation of microalgae for application in space.

Illumination with red and blue photons is known to be efficient for cultivation of higher plants. For microalgae cultivation, illumination with specific wavelengths rather than full spectrum illumination can be an alternative where there is a lack of knowledge about achievable biomass yields. This study deals with the usage of color LED illumination to cultivate microalgae integrated into closed life support systems for outer space. The goal is to quantify biomass yields using color illumination (red, blue, green and mixtures) compared to white light. Chlamydomonas reinhardtii was cultivated in plate reactors with color compared to white illumination regarding PCE, specific pigment concentration and cell size. Highest PCE values were achieved under low PFDs with a red/blue illumination (680 nm/447 nm) at a 90 to 10% molar ratio. At higher PFDs saturation effects can be observed resulting from light absorption characteristics and the linear part of PI curve. Cell size and aggregation are also influenced by the applied light color. Red/blue color illumination is a promising option applicable for microalgae-based modules of life support systems under low to saturating light intensities and double-sided illumination. Results of higher PCE with addition of blue photons to red light indicate an influence of sensory pigments.

Investigating the dynamics of recombinant protein secretion from a microalgal host.

Production of recombinant proteins with microalgae represents an alternative platform over plant- or bacterial-based expression systems for certain target proteins. Secretion of recombinant proteins allows accumulation of the target product physically separate from the valuable algal biomass. To date, there has been little investigation into the dynamics of recombinant protein secretion from microalgal hosts-the culture parameters that encourage secreted product accumulation and stability, while encouraging biomass production. In this work, the efficiency of recombinant protein production was optimized by adjusting cultivation parameters for a strain of Chlamydomonas reinhardtii previously engineered to secrete a functional recombinant Lolium perenne ice binding protein (LpIBP), which has applications as a frozen food texturing and cryopreservation additive, into its culture medium. Three media and several cultivation styles were investigated for effects on secreted LpIBP titres and culture growth. A combination of acetate and carbon dioxide feeding with illumination resulted in the highest overall biomass and recombinant protein titres up to 10mgL(-1) in the culture medium. Pure photoautotrophic production was possible using two media types, with recombinant protein accumulation in all cultivations correlating to culture cell density. Two different cultivation systems were used for scale-up to 10L cultivations, one of which produced yields of secreted recombinant protein up to 12mgL(-1) within six cultivation days. Functional ice recrystallization inhibition (IRI) of the LpIBP from total concentrated extracellular protein extracts was demonstrated in a sucrose solution used as a simplified ice cream model. IRI lasted up to 7 days, demonstrating the potential of secreted products from microalgae for use as food additives.

Biofuels from microalgae: photoconversion efficiency during lipid accumulation.

The accumulation of storage lipids in oleaginous microalgae can be induced by a targeted nutrient limitation. Experiments with varying concentrations of nitrate in the culture medium showed differing volumetric productivities of Phaeodactylum tricornutum in batch experiments. This was partially attributable to the differentiated ability of cultures to absorb light. Apart from that, it was demonstrated that storage molecule accumulation follows kinetics that show saturation at high photon flux densities. The measurement of the photoconversion efficiency (PCE) based on a rigorous balancing of absorbed light energy and changes in the enthalpy of combustion of biomass during nutrient depletion. In batch experiments the PCE was increased more than twofold, from 2.48% at low nitrate concentrations to a maximum value of 5.65%, by increase of the nitrogen availability.