Mild pressure induces rapid accumulation of neutral lipid (triacylglycerol) in Chlorella spp.

Effective enhancement of neutral lipid (especially triacylglycerol, TAG) content in microalgae is an important issue for commercialization of microalgal biorefineries. Pressure is a key physical factor affecting the morphological, physiological, and biochemical behaviors of organisms. In this paper, we report a new stress-based method for induction of TAG accumulation in microalgae (specifically, Chlorella sp. KR-1 and Ch. sp. AG20150) by very-short-duration application of mild pressure. Pressure treatments of 10-15bar for 2h resulted in a considerable, ∼55% improvement of the 10-100g/Lcells' TAG contents compared with the untreated control. The post-pressure-treatment increase of cytoplasmic TAG granules was further confirmed by transmission electron microscopy (TEM). Notwithstanding the increased TAG content, the total lipid content was not changed by pressurization, implying that pressure stress possibly induces rapid remodeling/transformation of algal lipids rather than de novo biosynthesis of TAG.

Magnesium aminoclay enhances lipid production of mixotrophic Chlorella sp. KR-1 while reducing bacterial populations.

Improving lipid productivity and preventing overgrowth of contaminating bacteria are critical issues relevant to the commercialization of the mixotrophic microalgae cultivation process. In this paper, we report the use of magnesium aminoclay (MgAC) nanoparticles for enhanced lipid production from oleaginous Chlorella sp. KR-1 with simultaneous control of KR-1-associated bacterial growth in mixotrophic cultures with glucose as the model substrate. Addition of 0.01-0.1g/L MgAC promoted microalgal biomass production better than the MgAC-less control, via differential biocidal effects on microalgal and bacterial cells (the latter being more sensitive to MgAC's bio-toxicity than the former). The inhibition effect of MgAC on co-existing bacteria was, as based on density-gradient-gel-electrophoresis (DGGE) analysis, largely dosage-dependent and species-specific. MgAC also, by inducing an oxidative stress environment, increased both the cell size and lipid content of KR-1, resulting in a considerable, ∼25% improvement of mixotrophic algal lipid productivity (to ∼410mgFAME/L/d) compared with the untreated control.

Efficient harvesting of wet blue-green microalgal biomass by two-aminoclay [AC]-mixture systems.

Blue-green microalgal blooms have been caused concerns about environmental problems and human-health dangers. For removal of such cyanobacteria, many mechanical and chemical treatments have been trialled. Among various technologies, the flocculation-based harvesting (precipitation) method can be an alternative if the problem of the low yield of recovered biomass at low concentrations of cyanobacteria is solved. In the present study, it was utilized mixtures of magnesium aminoclay [MgAC] and cerium aminoclay [CeAC] with different particle sizes to harvest cyanobacteria feedstocks with ∼100% efficiency within 1h by ten-fold lower loading of ACs compared with single treatments of [MgAC] or [CeAC]. This success was owed to the compact networks of the different-sized-ACs mixture for efficient bridging between microalgal cells. In order to determine the usage potential of biomass harvested with AC, the mass was heat treated under the reduction condition. This system is expected to be profitably utilizable in adsorbents and catalysts.

Magnetic-Nanoflocculant-Assisted Water-Nonpolar Solvent Interface Sieve for Microalgae Harvesting.

Exploitation of magnetic flocculants is regarded as a very promising energy-saving approach to microalgae harvesting. However, its practical applicability remains limited, mainly because of the problem of the postharvest separation of magnetic flocculants from microalgal flocs, which is crucial both for magnetic-flocculant recycling and high-purity microalgal biomasses, but which is also a very challenging and energy-consuming step. In the present study, we designed magnetic nanoflocculants dually functionalizable by two different organosilane compounds, (3-aminopropyl)triethoxysilane (APTES) and octyltriethoxysilane (OTES), which flocculate negatively charged microalgae and are readily detachable at the water-nonpolar organic solvent (NOS) interface only by application of an external magnetic field. APTES functionalization imparts a positive zeta potential charge (29.6 mV) to magnetic nanoflocculants, thereby enabling microalgae flocculation with 98.5% harvesting efficiency (with a dosage of 1.6 g of dMNF/g of cells). OTES functionalization imparts lipophilicity to magnetic nanoflocculants to make them compatible with NOS, thus effecting efficient separation of magnetic flocculants passing through the water-NOS interface sieve from hydrophilic microalgae. Our new energy-saving approach to microalgae harvesting concentrates microalgal cultures (∼1.5 g/L) up to 60 g/L, which can be directly connected to the following process of NOS-assisted wet lipid extraction or biodiesel production, and therefore provides, by simplifying multiple downstream processes, a great potential cost reduction in microalgae-based biorefinement.

Dramatic improvement of membrane performance for microalgae harvesting with a simple bubble-generator plate.

To overcome fouling issue in membrane-based algae harvesting and thus make an otherwise promising harvesting option more competitive, a bubble-generator plate was developed. According to computational fluid dynamics analysis, the plate generated substantial hydrodynamic power in terms of high pressure, velocity, and shear stress. When installed in a membrane filtration system with membranes of different surface and structural characteristics (one prepared by the phase inversion method, and a commercial one) the bubble-generator was indeed effective in reducing fouling. Without the plate, the much cheaper homemade membrane had the similar performance as the commercial one. Use of the bubble-generator considerably improved the performance of both membranes, and revealed a valuable synergy with the asymmetrical structure of the homemade membrane. This result clearly showed that the ever-problematic fouling could be mitigated in a rather easy manner, and in so doing, that membrane technology could indeed become a practical option for algae harvesting.

An integrated process for microalgae harvesting and cell disruption by the use of ferric ions.

In this study, a simultaneous process of harvesting biomass and extracting crude bio-oil was attempted from wet microalgae biomass using FeCl3 and Fe2(SO4)3 as both coagulant and cell-disrupting agent. A culture solution of Chlorella sp. KR-1 was firstly concentrated to 20 g/L and then proceeded for cell disruption with the addition of H2O2. Optimal dosage were 560 and 1060 mg/L for FeCl3 and Fe2(SO4)3, showing harvesting efficiencies of more than 99%. Optimal extraction conditions were identified via the response surface method (RSM), and the extraction yield was almost the same at 120 °C for both iron salts but FAME compositions after transesterification was found to be quite different. Given iron salts were a reference coagulant in water treatment in general and microalgae harvesting in particular, the present approach of using it for harvesting and oil-extraction in a simultaneous manner can serve as a practical route for the microalgae-derived biodiesel production.

Ferric chloride based downstream process for microalgae based biodiesel production.

In this study, ferric chloride (FeCl3) was used to integrate downstream processes (harvesting, lipid extraction, and esterification). At concentration of 200 mg/L and at pH 3, FeCl3 exhibited an expected degree of coagulation and an increase in cell density of ten times (170 mg/10 mL). An iron-mediated oxidation reaction, Fenton-like reaction, was used to extract lipid from the harvested biomass, and efficiency of 80% was obtained with 0.5% H2O2 at 90 °C. The iron compound was also employed in the esterification step, and converted free fatty acids to fatty acid methyl esters under acidic conditions; thus, the fatal problem of saponification during esterification with alkaline catalysts was avoided, and esterification efficiency over 90% was obtained. This study clearly showed that FeCl3 in the harvesting process is beneficial in all downstream steps and have a potential to greatly reduce the production cost of microalgae-originated biodiesel.

Improved biomass and lipid production in a mixotrophic culture of Chlorella sp. KR-1 with addition of coal-fired flue-gas.

Industrial CO2-rich flue-gases, owing to their eco-toxicity, have yet to be practically exploited for microalgal biomass and lipid production. In this study, various autotrophic and mixotrophic culture modes for an oleaginous microalga, Chlorella sp. KR-1 were compared for the use in actual coal-fired flue-gas. Among the mixotrophic conditions tested, the fed-batch feedings of glucose and the supply of air in dark cycles showed the highest biomass (561 mg/L d) and fatty-acid methyl-ester (168 mg/L d) productivities. This growth condition also resulted in the maximal population of microalgae and the minimal population and types of KR-1-associated-bacterial species as confirmed by particle-volume-distribution and denaturing-gradient-gel-electrophoresis (DGGE) analyses. Furthermore, microalgal lipid produced was assessed, based on its fatty acid profile, to meet key biodiesel standards such as saponification, iodine, and cetane numbers.

Repeated use of stable magnetic flocculant for efficient harvest of oleaginous Chlorella sp.

In the present study, a simple magnetic-particle recycling strategy was developed for harvest of the oleaginous microalga Chlorella sp. KR-1. The method entails the flocculation of microalgal cells and bare-Fe3O4 magnetic particles (bMP) by electrostatic attraction and the subsequent recovery of the bMP from the harvested flocs by electrostatic repulsion below and above the isoelectric points (IEP), respectively. For 10 recycles, the bMP showed 94-99% and 90-97% harvest and recovery efficiencies, respectively. Furthermore, neither the use of bMP nor pH adjustment showed any adverse effect on the microalgal cell growth or the co-existing bacterial species, as confirmed from the subsequent medium-recycling test and denaturing gradient gel electrophoresis (DGGE) analysis.

Mixotrophic cultivation of oleaginous Chlorella sp. KR-1 mediated by actual coal-fired flue gas for biodiesel production.

Flue gases mainly consist of CO2 that can be utilized to facilitate microalgal culture for bioenergy production. In the present study, to evaluate the feasibility of the utilization of flue gas from a coal-burning power plant, an indigenous and high-CO2-tolerant oleaginous microalga, Chlorella sp. KR-1, was cultivated under mixotrophic conditions, and the results were evaluated. When the culture was mediated by flue gas, highest biomass (0.8 g cells/L·d) and FAME (fatty acid methyl esters) productivity (121 mg/L·d) were achieved in the mixotrophic mode with 5 g/L glucose, 5 mM nitrate, and a flow rate of 0.2 vvm. By contrast, the photoautotrophic cultivation resulted in a lower biomass (0.45 g cells/L·d) and a lower FAME productivity (60.2 mg/L·d). In general, the fatty acid profiles of Chlorella sp. KR-1 revealed meaningful contents (>40 % of saturated and mono-unsaturated fatty acids) under the mixotrophic condition, which enables the obtainment of a better quality of biodiesel than is possible under the autotrophic condition. Conclusively then, it was established that a microalgal culture mediated by flue gas can be improved by adoption of mixotrophic cultivation systems.

Aquatic ecotoxicity effect of engineered aminoclay nanoparticles.

In the present study the short term aquatic ecotoxicity of water-solubilized aminoclay nanoparticles (ANPs) of ~51±31 nm average hydrodynamic diameter was characterized. An ecotoxicological evaluation was carried out utilizing standard test organisms of different phyla and trophic levels namely the eukaryotic microalga Pseudokirchneriella subcapitata, the crustacean Daphnia magna and the bioluminescent marine bacteria Vibrio fisheri. The effective inhibitory concentration (EC50) with 95% confidence limits for the microalga was 1.29 mg/L (0.72-1.82) for the average growth rate and 0.26 mg/L (0.23-0.31) for the cell yield. The entrapping of algal cells in aggregates of ANP may play a major role in the growth inhibition of algae P. subcapitata. No inhibition was observed for V. fisheri up to 25,000 mg/L (no observed effect concentration; NOEC). For D. magna no immobilization was observed in a limit test with 100 mg/L in 24 h while in 48 h a single animal was immobilized (5% inhibition). Correspondingly, the NOEC of ANP in 24 h was 100 mg/L and the lowest observed effect concentration (LOEC) for 48 h was 100 mg/L. Therefore it can be considered to use ANP as an algal-inhibition agent at concentrations <100 mg/L without affecting or only mildly affecting other organisms including zooplanktons, but further studies on the environmental fate and chronic toxicity of ANP is needed to confirm this.

Aminoclay-induced humic acid flocculation for efficient harvesting of oleaginous Chlorella sp.

Biofuels (biodiesel) production from oleaginous microalgae has been intensively studied for its practical applications within the microalgae-based biorefinement process. For scaled-up cultivation of microalgae in open ponds or, for further cost reduction, using wastewater, humic acids present in water-treatment systems can positively and significantly affect the harvesting of microalgae biomass. Flocculation, because of its simplicity and inexpensiveness, is considered to be an efficient approach to microalgae harvesting. Based on the reported cationic aminoclay usages for a broad spectrum of microalgae species in wide-pH regimes, aminoclay-induced humic acid flocculation at the 5g/L aminoclay loading showed fast floc formation, approximately 100% harvesting efficiency, which was comparable to the only-aminoclay treatment at 5g/L, indicating that the humic acid did not significantly inhibit the microalgae harvesting behavior. As for the microalgae flocculation mechanism, it is suggested that cationic nanoparticles decorated on macromolecular matters function as a type of network in capturing microalgae.

Harvesting of oleaginous Chlorella sp. by organoclays.

In microalgae-based biorefinement, one of the highest practical priorities is to reduce the costs of downstream processes. As one potential solution, microalgae harvesting by organoclays has received particularly keen research interest. In the present study, cationic charged aluminum- and magnesium-backboned organoclays were synthesized and solubilized in aqueous solution due to their high-density of amino sites. Each, within 30 min of its injection into 1.7 g/L-concentration microalgal feedstocks, effected harvesting efficiencies of almost 100% at concentrations above 0.6 g/L while maintaining a neutral pH. Conclusively, organoclays, if recycled efficiently, can be uniquely effective microalgae harvesting agents.

Overexpression of FTL1/DDF1, an AP2 transcription factor, enhances tolerance to cold, drought, and heat stresses in Arabidopsis thaliana.

Freezing temperatures control where and when plants can grow, and negatively influence crop quality and productivity. To identify key regulatory genes involved in cold adaptation, we screened activation-tagged Arabidopsis lines for mutants with greater freezing tolerance. One mutant, freezing tolerant line1 (ftl1-1D), manifested enhanced tolerance along with dwarfism and delayed flowering. This was caused by activation of DWARF AND DELAYED FLOWERING 1 (DDF1), a gene previously described as a regulatory component in salinity signaling. The induced gene encoded an AP2 transcription factor of the CBF/DREB1 subfamily. In addition to conferring tolerance to low temperatures and salt stress, ftl1-1D/ddf1 improved tolerance to drought and heat. Real-time PCR indicated that FTL1/DDF1 was up-regulated by those four types of stresses in wild-type Arabidopsis. Its increased expression in the mutant induced various stress-responsive genes under normal growing conditions, resulting in improved tolerances. However, phenotypes shown in the ftl1-1D/ddf1 were restored by treatment with exogenous gibberellin (GA3), indicating the involvement of a GA pathway in FTL1/DDF1-mediated tolerance. Therefore, we conclude that FTL1/DDF1 plays a role in regulating responses to several abiotic stresses, perhaps via cross-talk in the pathways.