Harvesting of marine microalgae using cationic cellulose nanocrystals.

Flocculation of marine microalgae is challenging because of the high ionic strength of the culture medium. We investigated cationic cellulose nanocrystals (CNCs) as biobased flocculants for the marine microalgae Nannochloropsis oculata, and compared its performance to chitosan. Cationic CNCs induced flocculation at a low dose of 11 mg L-1, while chitosan required a dose of 35 mg L-1. Our cationic CNCs possess a permanent positive charge, allowing flocculation over a wide pH-range (4 to 10). The CNC maximum flocculation efficiency was 90 %, while chitosan achieved > 95 %, attributed to small flocs remaining in suspension for CNCs. However, centrifugation for 1 min at 180 g or gravity filtration using a 30 µm nylon filter after CNC flocculation resulted in the removal of these small, stable flocs (∼150 µm diam) and > 95% harvesting efficiency. Cationic CNCs can thus serve as a sustainable alternative natural flocculant for harvesting both freshwater and marine microalgae.

Anaerobic batch conversion of pine wood torrefaction condensate.

Organic compound rich torrefaction condensate, owing to their high water content and acidic nature, have yet to be exploited for practical application. In this study, microbial conversion of torrefaction condensate from pine wood through anaerobic batch digestion (AD) to produce methane was evaluated. Torrefaction condensate exhibited high methane potentials in the range of 430-492mL/g volatile solids (VS) and 430-460mL/gVS under mesophilic and thermophilic conditions, respectively. Owing to the changes in the composition, the methane yields differed with the torrefaction condensates produced at different temperatures (225, 275 and 300°C), with a maximum of 492±18mL/gVS with the condensate produced at 300°C under mesophilic condition. The cyclic batch AD experiments showed that 0.1VSsubstrate:VSinoculum is optimum, whereas the higher substrate loading (0.2-0.5) resulted in a reversible inhibition of the methane production. The results suggest that torrefaction condensate could be practically valorized through AD.

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.

Cell-wall disruption and lipid/astaxanthin extraction from microalgae: Chlorella and Haematococcus.

Recently, biofuels and nutraceuticals produced from microalgae have emerged as major interests, resulting in intensive research of the microalgal biorefinery process. In this paper, recent developments in cell-wall disruption and extraction methods are reviewed, focusing on lipid and astaxanthin production from the biotechnologically important microalgae Chlorella and Haematococcus, respectively. As a common, critical bottleneck for recovery of intracellular components such as lipid and astaxanthin from these microalgae, the composition and structure of rigid, thick cell-walls were analyzed. Various chemical, physical, physico-chemical, and biological methods applied for cell-wall breakage and lipid/astaxanthin extraction from Chlorella and Haematococcus are discussed in detail and compared based on efficiency, energy consumption, type and dosage of solvent, biomass concentration and status (wet/dried), toxicity, scalability, and synergistic combinations. This report could serve as a useful guide to the implementation of practical downstream processes for recovery of valuable products from microalgae including Chlorella and Haematococcus.

Alginate microgels created by selective coalescence between core drops paired with an ultrathin shell.

We report a highly biocompatible and practical protocol to create alginate microgels for bioactive encapsulation. Double-emulsion drops composed of dual cores enclosed by an ultrathin shell are prepared in a capillary microfluidic device, which exhibit selective coalescence between the cores. When the cores are laden with alginate precursors and divalent ions, respectively, coalescence leads to the formation of alginate microgels in the fused core of double-emulsion drops. The microgel can be rapidly released into a continuous water phase by rupturing the liquid shell. This method neither involves any toxic chemical cues for gelation nor long-term exposure to oil, thereby providing highly biocompatible encapsulation.

Regenerative Astaxanthin Extraction from a Single Microalgal (Haematococcus pluvialis) Cell Using a Gold Nano-Scalpel.

Milking of microalgae, the process of reusing the biomass for continuous production of target compounds, can strikingly overcome the time and cost constraints associated with biorefinery. This process can significantly improve production efficiency of highly valuable chemicals, for example, astaxanthin (AXT) from Haematococcus pluvialis. Detailed understanding of the biological process of cell survival and AXT reaccumulation after extraction would be of great help for successful milking. Here we report extraction of AXT from a single cell of H. pluvialis through incision of the cell wall by a gold nanoscalpel (Au-NS), which allows single-cell analysis of wound healing and reaccumulation of AXT. Interestingly, upon the Au-NS incision, the cell could reaccumulate AXT at a rate two times faster than the control cells. Efficient extraction as well as minimal cellular damage, keeping cells alive, could be achieved with the optimized shape and dimensions of Au-NS: a well-defined sharp tip, thickness under 300 nm, and 1-3 µm of width. The demonstration of regenerative extraction of AXT at a single cell level hints toward the potential of a milking process for continuous recovery of target compounds from microalgae while keeping the cells alive.

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.

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.

Magnetophoretic harvesting of oleaginous Chlorella sp. by using biocompatible chitosan/magnetic nanoparticle composites.

The consumption of energy and resources such as water in the cultivation and harvesting steps should be minimized to reduce the overall cost of biodiesel production from microalgae. Here we present a biocompatible and rapid magnetophoretic harvesting process of oleaginous microalgae by using chitosan-Fe3O4 nanoparticle composites. Over 99% of microalgae was harvested by using the composites and the external magnetic field without changing the pH of culture medium so that it may be reused for microalgal culture without adverse effect on the cell growth. Depending on the working volume (20-500 mL) and the strength of surface magnetic-field (3400-9200 G), the process of harvesting microalgae took only 2-5 min. The method presented here not only utilizes permanent magnets without additional energy for fast harvesting but also recycles the medium effectively for further cultivation of microalgae, looking ahead to a large scale economic microalgae-based biorefinement.

Biochemical changes of fresh water cyanobacteria Dolichospermum flos-aquae NTMS07 to chromium-induced stress with special reference to antioxidant enzymes and cellular fatty acids.

This investigation examined the efficiency of Dolichospermum flos-aquae NTMS07 in the removal of Cr(VI) from exposure water at various concentrations (2.5, 5, 7.5, 10 mg/L) over different time intervals of contact (1-5 days). Chromium removal was maximum at 2.5 mg/L, and decreased with increased concentration. The responses of the antioxidative enzymes superoxide dismutase (SOD) and catalase (CAT) were measured, and the composition of fatty acids was evaluated at a concentration of 5 mg Cr/L. Significant increases in the activity levels of SOD and CAT were obtained. The level of total unsaturated fatty acids decreased with exposure to Cr. It is proposed that the observed decrease in total unsaturated fatty acid level is a defense mechanism against Cr-induced oxidative stress and cell membrane damage.

Oxidative stress response and fatty acid changes associated with bioaccumulation of chromium [Cr(VI)] by a fresh water cyanobacterium Chroococcus sp.

Cr(VI) at 2.5, 5, 7.5 and 10 mg/l was removed over 1-5 days by a freshwater cyanobacterium, Chroococcus sp. 2.5 mg Cr(VI)/l gave the optimum rate. With 5 mg Cr(VI)/l, activities of superoxide dismutase and catalase were increased. Amounts of palmitic (16:0), stearic (18:0) and oleic acid (18:1) in the cell also increased after exposure to Cr(VI).