Biofilm growth of Chlorella sorokiniana in a rotating biological contactor based photobioreactor.

Microalgae biofilms could be used as a production platform for microalgae biomass. In this study, a photobioreactor design based on a rotating biological contactor (RBC) was used as a production platform for microalgae biomass cultivated in biofilm. In the photobioreactor, referred to as Algadisk, microalgae grow in biofilm on vertical rotating disks partially submerged in a growth medium. The objective is to evaluate the potential of the Algadisk photobioreactor with respect to the effects of disk roughness, disk rotation speed and CO2 concentration. These objectives where evaluated in relationship to productivity, photosynthetic efficiency, and long-term cultivation stability in a lab-scale Algadisk system. Although the lab-scale Algadisk system is used, operation parameters evaluated are relevant for scale-up. Chlorella Sorokiniana was used as model microalgae. In the lab-scale Algadisk reactor, productivity of 20.1 ± 0.7 g per m(2) disk surface per day and a biomass yield on light of 0.9 ± 0.04 g dry weight biomass per mol photons were obtained. Different disk rotation speeds did demonstrate minimal effects on biofilm growth and on the diffusion of substrate into the biofilm. CO2 limitation, however, drastically reduced productivity to 2-4 g per m(2) disk surface per day. Productivity could be maintained over a period of 21 weeks without re-inoculation of the Algadisk. Productivity decreased under extreme conditions such as pH 9-10, temperature above 40°C, and with low CO2 concentrations. Maximal productivity, however, was promptly recovered when optimal cultivation conditions were reinstated. These results exhibit an apparent opportunity to employ the Algadisk photobioreactor at large scale for microalgae biomass production if diffusion does not limit the CO2 supply.

Mutations in the M-gene segment can substantially increase replication efficiency of NS1 deletion influenza A virus in MDCK cells.

Influenza viruses unable to express NS1 protein (delNS1) replicate poorly and induce large amounts of interferon (IFN). They are therefore considered candidate viruses for live-attenuated influenza vaccines. Their attenuated replication is generally assumed to result from the inability to counter the antiviral host response, as delNS1 viruses replicate efficiently in Vero cells, which lack IFN expression. In this study, delNS1 virus was parallel passaged on IFN-competent MDCK cells, which resulted in two strains that were able to replicate to high virus titers in MDCK cells due to adaptive mutations especially in the M-gene segment but also in the NP and NS gene segments. Most notable were clustered U-to-C mutations in the M segment of both strains and clustered A-to-G mutations in the NS segment of one strain, which presumably resulted from host cell-mediated RNA editing. The M segment mutations in both strains changed the ratio of M1 to M2 expression, probably by affecting splicing efficiency. In one virus, 2 amino acid substitutions in M1 additionally enhanced virus replication, possibly through changes in the M1 distribution between the nucleus and the cytoplasm. Both adapted viruses induced levels of IFN equal to that of the original delNS1 virus. These results show that the increased replication of the adapted viruses is not primarily due to altered IFN induction but rather is related to changes in M1 expression or localization. The mutations identified in this paper may be used to enhance delNS1 virus replication for vaccine production.

Impact of hydraulic retention time on community assembly and function of photogranules for wastewater treatment.

Photogranules are dense, spherical agglomerates of cyanobacteria, microalgae and non-phototrophic microorganisms that have considerable advantages in terms of harvesting and nutrient removal rates for light driven wastewater treatment processes. This ecosystem is poorly understood in terms of the microbial community structure and the response of the community to changing abiotic conditions. To get a better understanding, we investigated the effect of hydraulic retention time (HRT) on photogranule formation and community assembly over a period of 148 days. Three laboratory bioreactors were inoculated with field samples from various locations in the Netherlands and operated in sequencing batch mode. The bioreactors were operated at four different HRTs (2.00, 1.00, 0.67, 0.33 days), while retaining the same solid retention time of 7 days. A microbial community with excellent settling characteristics (95-99% separation efficiency) was established within 2-5 weeks. The observed nutrient uptake rates ranged from 24 to 90 mgN L-1 day-1 and from 3.1 to 5.4 mgP L-1 day-1 depending on the applied HRT. The transition from single-cell suspension culture to floccular agglomeration to granular sludge was monitored by microscopy and 16S/18S sequencing. In particular, two important variables for driving aggregation and granulation, and for the structural integrity of photogranules were identified: 1. Extracellular polymeric substances (EPS) with high protein to polysaccharide ratio and 2. specific microorganisms. The key players were found to be the cyanobacteria Limnothrix and Cephalothrix, the colony forming photosynthetic eukaryotes within Chlamydomonadaceae, and the biofilm producing bacteria Zoogloea and Thauera. Knowing the makeup of the microbial community and the operational conditions influencing granulation and bioreactor function is crucial for successful operation of photogranular systems.

Cell kinetics of the marine sponge Halisarca caerulea reveal rapid cell turnover and shedding.

This study reveals the peculiar in vivo cell kinetics and cell turnover of the marine sponge Halisarca caerulea under steady-state conditions. The tropical coral reef sponge shows an extremely high proliferation activity, a short cell cycle duration and massive cell shedding. Cell turnover is predominantly confined to a single cell population, i.e. the choanocytes, and in this process apoptosis only plays a minor role. To our knowledge, such fast cell kinetics under steady-state conditions, with high turnover by shedding in the absence of apoptosis, has not been observed previously in any other multicellular organism. The duration of the cell cycle in vivo resembles that of unicellular organisms in culture. Morphological and histochemical studies demonstrate compartmentalization of choanocytes in the sponge tissue, which corresponds well with its remarkable cellular kinetics. Coral reef cavity sponges, like H. caerulea, inhabit low nutrient tropical waters, forcing these organisms to filter large volumes of water and to capture the few nutrients efficiently. Under these oligotrophic conditions, a high cell turnover may be considered as a very useful strategy, preventing permanent damage to the sponge by environmental stress. Halisarca caerulea maintains its body mass and keeps its food uptake system up to date by constantly renewing its filter system. We conclude that studies on cell kinetics and functional morphology provide new and essential information on the growth characteristics and the regulation of sponge growth in vivo as well as in vitro and the role of choanocytes in tissue homeostasis.

Growth inhibition of Monodus subterraneus by free fatty acids.

Monodus subterraneus is a microalga, which is known for its high eicosapentaenoic acid (EPA; 20:5omega3) content. To produce EPA commercially, high volumetric productivities of microalgae are required. These high productivities can be reached in flat panel photobioreactors with small optical paths that have to be operated at high cell densities (>10 g/L). However, at these cell densities a reduction of productivity is observed. This growth inhibition is probably caused by growth inhibitors released by the microalgae, which have been suggested to be fatty acids. Our aim was to investigate if free fatty acids produced by M. subterraneus inhibited growth of this species. Therefore a bioassay was developed and saturated, unsaturated and poly-unsaturated fatty acids occurring in Monodus were tested on their growth inhibiting properties. Growth of M. subterraneus was completely inhibited at a saturated concentration (96 microM) of palmitoleic acid (16:1omega7). But, the saturated fatty acid palmitic acid (16:0) and the mono-saturated oleic acid (18:1omega9) were much stronger inhibitors. Growth was inhibited for 50% already at concentrations of 0.4 microM 16:0 and 3 microM 18:1omega9, respectively. These fatty acids probably cause the growth inhibition in high cell density cultures of M. subterraneus.

Cell cultures from the symbiotic soft coral Sinularia flexibilis.

The symbiotic octocoral Sinularia flexibilis is a producer of potential pharmaceuticals. Sustainable mass production of these corals as a source of such compounds demands innovative approaches, including coral cell culture. We studied various cell dissociation methodologies and the feasibility of cultivation of S. flexibilis cells on different media and cell dissociation methodologies. Mechanical dissociation of coral tissue always yielded the highest number of cells and allowed subsequent cellular growth in all treatments. The best results from chemical dissociation reagents were found with trypsin-ethylene diamine tetraacetic acid. Coral cells obtained from spontaneous dissociation did not grow. Light intensity was found to be important for coral cell culture showing an enduring symbiosis between the cultured cells and their intracellular algae. The Grace's insect medium and Grace's modified insect medium were found to be superior substrates. To confirm the similarity of the cultured cells and those in the coral tissue, a molecular test with Internal Transcribed Spacer primers was performed. Thereby, the presence of similar cells of both the coral cells and zooxanthella in different culture media was confirmed.

Multi-Product Microalgae Biorefineries: From Concept Towards Reality.

Although microalgae are a promising biobased feedstock, industrial scale production is still far off. To enhance the economic viability of large-scale microalgae processes, all biomass components need to be valorized, requiring a multi-product biorefinery. However, this concept is still too expensive. Typically, downstream processing of industrial biotechnological bulk products accounts for 20-40% of the total production costs, while for a microalgae multi-product biorefinery the costs are substantially higher (50-60%). These costs are high due to the lack of appropriate and mild technologies to access the different product fractions such as proteins, carbohydrates, and lipids. To reduce the costs, simplified processes need to be developed for the main unit operations including harvesting, cell disruption, extraction, and possibly fractionation.

Selective and energy efficient extraction of functional proteins from microalgae for food applications.

The use of a single controlled bead milling step of the microalga Tetraselmis suecica resulted in a soluble fraction, rich in functional proteins. This was achieved by fine-tuning the processing time, thereby exploiting the difference in rates of protein and carbohydrate release during milling. Soluble proteins were extracted under mild conditions -room temperature, no addition of chemicals, pH 6.5-, with a yield of 22.5% and a specific energy consumption of 0.6 kWh kgDW-1, which is within the recommended minimum energy for an extraction step in a biorefinery process. The resulting protein extract contained 50.4% (DW) of proteins and 26.4% carbohydrates, showed light green color and displayed superior surface activity and gelation behavior compared to whey protein isolate. The proposed process is simple (only one bead milling step), scalable, and allows the mild extraction of functional proteins, making it interesting for industrial applications in the food industry.

Techno-Functional Properties of Crude Extracts from the Green Microalga Tetraselmis suecica.

A mild fractionation process to extract functional biomolecules from green microalgae was implemented. The process includes bead milling, centrifugation, and filtration with several membrane cut-offs. For each fraction, the corresponding composition was measured, and the surface activity and gelation behavior were determined. A maximum protein yield of 12% was obtained in the supernatant after bead milling and between 3.2 and 11.7% after filtration. Compared to whey protein isolate, most of the algae fractions exhibited comparable or enhanced functionality. Surface activity for air-water and oil-water interfaces and gelation activities were notably superior for the retentate fractions compared to the permeates. It is proposed that such functionality in the retentates is due to the presence of hydrophobic compounds and molecular complexes exhibiting a similar behavior as Pickering particles. We demonstrated that excellent functionality can be obtained with crude fractions, requiring minimum processing and, thus, constituting an interesting option for commercial applications.

Biorefinery of the macroalgae Ulva lactuca: extraction of proteins and carbohydrates by mild disintegration.

The effect of osmotic shock, enzymatic incubation, pulsed electric field, and high shear homogenization on the release of water-soluble proteins and carbohydrates from the green alga Ulva lactuca was investigated in this screening study. For osmotic shock, both temperature and incubation time had a significant influence on the release with an optimum at 30 °C for 24 h of incubation. For enzymatic incubation, pectinase demonstrated being the most promising enzyme for both protein and carbohydrate release. Pulsed electric field treatment was most optimal at an electric field strength of 7.5 kV cm-1 with 0.05 ms pulses and a specific energy input relative to the released protein as low as 6.6 kWh kgprot-1. Regarding literature, this study reported the highest protein (~ 39%) and carbohydrate (~ 51%) yields of the four technologies using high shear homogenization. Additionally, an energy reduction up to 86% was achieved by applying a novel two-phase (macrostructure size reduction and cell disintegration) technique.

Gene silencing of stearoyl-ACP desaturase enhances the stearic acid content in Chlamydomonas reinhardtii.

In this study, stearoyl-ACP desaturase (SAD), the enzyme that converts stearic acid into oleic acid, is silenced by artificial microRNA in the green microalga Chlamydomonas reinhardtii. Two different constructs, which target different positions on the mRNA of stearoyl-ACP desaturase, were tested. The mRNA levels for SAD were reduced after the silencing construct was induced. In one of the strains, the reduction in SAD mRNA resulted in a doubling of the stearic acid content in triacylglycerol molecules, which shows that stearic acid production in microalgae is possible.

Continuous versus batch production of lipids in the microalgae Acutodesmus obliquus.

This work provides a novel quantitative comparison of batch versus continuous microalgal lipid production in the wild type and starchless mutant strain of Acutodesmus obliquus. Both strains showed higher TAG yields on light under batch operation compared to continuous nitrogen limitation. The starchless mutant showed 0.20gTAGmolph-1 for batch and 0.12gTAGmolph-1 for continuous operation, while the wildtype only showed 0.16gTAGmolph-1 for batch and 0.08gTAGmolph-1 for continuous operation. Also, higher TAG contents were found under batch starvation (26% of dry weight for the wildtype and 43% of dry weight for starchless mutant) compared to continuous cultivations (16% of dry weight for the wildtype and 33% of dry weight for starchless mutant). Starch acts as the favoured storage metabolite during nitrogen limitation in A. obliquus, whereas TAG is only accumulated after starch reaches a cellular maximum of 40% of dry weight.

Energy efficient bead milling of microalgae: Effect of bead size on disintegration and release of proteins and carbohydrates.

The disintegration of three industry relevant algae (Chlorella vulgaris, Neochloris oleoabundans and Tetraselmis suecica) was studied in a lab scale bead mill at different bead sizes (0.3-1mm). Cell disintegration, proteins and carbohydrates released into the water phase followed a first order kinetics. The process is selective towards proteins over carbohydrates during early stages of milling. In general, smaller beads led to higher kinetic rates, with a minimum specific energy consumption of ⩽0.47kWhkgDW-1 for 0.3mm beads. After analysis of the stress parameters (stress number and stress intensity), it appears that optimal disintegration and energy usage for all strains occurs in the 0.3-0.4mm range. During the course of bead milling, the native structure of the marker protein Rubisco was retained, confirming the mildness of the disruption process.

Draft Genome Sequence of the Oleaginous Green Alga Tetradesmus obliquus UTEX 393.

The microalgae Tetradesmus obliquus is able to maintain a high photosynthetic efficiency under nitrogen limitation and is considered a promising green microalgae for sustainable production of diverse compounds, including biofuels. Here, we report the first draft whole-genome shotgun sequencing of T. obliquus The final assembly comprises 108,715,903 bp with over 1,368 scaffolds.

Understanding the salinity effect on cationic polymers in inducing flocculation of the microalga Neochloris oleoabundans.

A mechanistic study was performed to evaluate the effect of salinity on cationic polymeric flocculants, that are used for the harvesting of microalgae. The polyacrylamide Synthofloc 5080H and the polysaccharide Chitosan were employed for the flocculation of Neochloris oleoabundans. In seawater conditions, a maximum biomass recovery of 66% was obtained with a dosage of 90mg/L Chitosan. This recovery was approximately 25% lower compared to Synthofloc 5080H reaching recoveries greater than 90% with dosages of 30mg/L. Although different recoveries were obtained with both flocculants, the polymers exhibit a similar apparent polymer length, as was evaluated from viscosity measurements. While both flocculants exhibit similar polymer lengths in increasing salinity, the zeta potential differs. This indicates that polymeric charge dominates flocculation. With increased salinity, the effectivity of cationic polymeric flocculants decreases due to a reduction in cationic charge. This mechanism was confirmed through a SEM analysis and additional experiments using flocculants with various charge densities.

Selective extraction of intracellular components from the microalga Chlorella vulgaris by combined pulsed electric field-temperature treatment.

The synergistic effect of temperature (25-65 °C) and total specific energy input (0.55-1.11 kWh kgDW(-1)) by pulsed electric field (PEF) on the release of intracellular components from the microalgae Chlorella vulgaris was studied. The combination of PEF with temperatures from 25 to 55 °C resulted in a conductivity increase of 75% as a result of cell membrane permeabilization. In this range of temperatures, 25-39% carbohydrates and 3-5% proteins release occurred and only for carbohydrate release a synergistic effect was observed at 55 °C. Above 55 °C spontaneous cell lysis occurred without PEF. Combined PEF-temperature treatment does not sufficiently disintegrate the algal cells to release both carbohydrates and proteins at yields comparable to the benchmark bead milling (40-45% protein, 48-58% carbohydrates).

Logistic analysis of algae cultivation.

Energy requirements for resource transport of algae cultivation are unknown. This work describes the quantitative analysis of energy requirements for water and CO2 transport. Algae cultivation models were combined with the quantitative logistic decision model 'BeWhere' for the regions Benelux (Northwest Europe), southern France and Sahara. For photobioreactors, the energy consumed for transport of water and CO2 turns out to be a small percentage of the energy contained in the algae biomass (0.1-3.6%). For raceway ponds the share for transport is higher (0.7-38.5%). The energy consumption for transport is the lowest in the Benelux due to good availability of both water and CO2. Analysing transport logistics is still important, despite the low energy consumption for transport. The results demonstrate that resource requirements, resource distribution and availability and transport networks have a profound effect on the location choices for algae cultivation.

Dosage effect of cationic polymers on the flocculation efficiency of the marine microalga Neochloris oleoabundans.

A mechanistic mathematical model was developed to predict the performance of cationic polymers for flocculating salt water cultivated microalgae. The model was validated on experiments carried out with Neochloris oleoabundans and three different commercial flocculants (Zetag 7557®, Synthofloc 5080H® and SNF H536®). For a wide range of biomass concentrations (0.49-1.37 g L(-1)) and flocculant dosages (0-150 mg L(-1)) the model simulations predicted well the optimal flocculant-to-biomass ratio between 43 and 109 mgflocculant/gbiomass. At optimum conditions biomass recoveries varied between 88% and 99%. The cost of the usage of commercial available flocculants is estimated to range between 0.15$/kgbiomass and 0.49$/kgbiomass.

Mild disintegration of the green microalgae Chlorella vulgaris using bead milling.

In this work, the mild disintegration of the microalgae Chlorella vulgaris for the release of intracellular products has been studied. By means of bead milling the microalgae suspensions were successfully disintegrated at different biomass concentrations (25-145 gDW kg(-1)) over a range of agitator speeds (6-12 m s(-1)). In all cases over 97% of cell disintegration was achieved resulting in a release of water soluble proteins. A clear optimum rate of disintegration and protein release was observed at an agitator speed of 9-10 m s(-1) regardless of the biomass concentration. Selective extraction of water soluble proteins was observed as proteins released sooner than cell disintegration took place. Proteins could be released at 85% lower energy input than for cell disintegration resulting in specific energy consumptions well below 2.5 kWh kgDW(-1).

Cell disruption for microalgae biorefineries.

Microalgae are a potential source for various valuable chemicals for commercial applications ranging from nutraceuticals to fuels. Objective in a biorefinery is to utilize biomass ingredients efficiently similarly to petroleum refineries in which oil is fractionated in fuels and a variety of products with higher value. Downstream processes in microalgae biorefineries consist of different steps whereof cell disruption is the most crucial part. To maintain the functionality of algae biochemicals during cell disruption while obtaining high disruption yields is an important challenge. Despite this need, studies on mild disruption of microalgae cells are limited. This review article focuses on the evaluation of conventional and emerging cell disruption technologies, and a comparison thereof with respect to their potential for the future microalgae biorefineries. The discussed techniques are bead milling, high pressure homogenization, high speed homogenization, ultrasonication, microwave treatment, pulsed electric field treatment, non-mechanical cell disruption and some emerging technologies.