Development of a new sterilization method for microalgae media using calcium hypochlorite as the sterilant.

Calcium hypochlorite (Ca(ClO)2), which can be stably stored in powder form for a long period, is widely used as a disinfectant in various fields. A new sterilization process was developed in the present study, where a microalgal medium was sterilized using 0.02% Ca(ClO)2, followed by complete neutralization of the Ca(ClO)2 within 8 h through catalytic reaction of an MnCl2-Na2EDTA complex and a synergistic effect of glucose. When comparing the growth of Chlorella vulgaris in the autoclaved medium, a 2.65 times greater maximum cell growth was observed in cells grown in the medium prepared by treatment of Ca(ClO)2. This result indicates that denaturation of the medium by heat can hinder the growth of some microorganisms. In the case of cultivation of Euglena gracilis, successful culture growth was achieved without growth inhibition or contamination on a medium prepared in the same manner.

Photometric determination of peracetic acid by reaction with potassium iodide solution.

Peracetic acid (PAA) is a strong oxidizing agent and is considered an ideal disinfectant because of its excellent disinfecting effect at low concentration, low corrosiveness, and relatively low cost. Commercially available PAA solution is a mixture of PAA, acetic acid, and hydrogen peroxide. However, PAA naturally decomposes faster than hydrogen peroxide. Therefore, accurately quantifying the concentration of PAA in the PAA peroxide mixture via a simple method is important. In the present study, a new method was developed, in which the spectral change of I- ion at 226 nm and the absorption value from the generated I2 at 460 nm were used to determine the concentration of PAA, following a chemical reaction with 0.1 mM potassium iodide (KI) solution without the use of any other chemicals. In this work, the measurable concentration of PAA was as low as 0.0001 wt% (13.1 µM) and as high as 0.0015 wt% (197.2 µM), which matches well with high linearity (99.95% at 226 nm and 99.91% at 460 nm). This work could also be the high selectivity method toward PAA in the PAA peroxide mixture.

A simple spectrophotometric determination of hydrogen peroxide solution via spectral delta from redox reaction with sodium hypochlorite.

Hydrogen peroxide (H2O2) is widely used in the synthesis of organic chemicals, bleaching of paper pulp, and the treatment of wastewater and as a food additive, important mediator of redox processes in natural water, and a disinfectant. However, H2O2 stock solution is unstable and slowly decomposes when exposed to, for example, light, elevated temperatures, or metal compounds. Therefore, the ability to measure the exact concentration of H2O2 stock solution is important for its proper use in diverse applications. This work proposes a simple method for the spectrophotometric determination of H2O2 solution via chemical reaction with sodium hypochlorite that is inexpensive and easy to acquire. The proposed method is based on the stoichiometric spectral change of hypochlorite ion at 292.5 nm following a redox reaction with a sample solution of H2O2. Due to high relationship between the spectral delta value and the applied H2O2 concentration (0.00188-0.03000%), H2O2 stock solution can be easily quantified.

A simple method for extracting phycocyanin from Arthrospira (Spirulina) platensis by autolysis.

Phycocyanin (PC) is a natural blue pigment that has great commercial value in food and pharmaceutical industry. Arthrospira (Spirulina) platensis is a photosynthetic spiral-shaped cyanobacterium containing a rich PC pigment. Autolysis is the enzymatic digestion of cells by the action of its own enzymes. To develop an effective and economical extraction process, an autolysis process was incorporated into the conventional freezing-thawing method. In the present study, 91% of maximal extraction yield of PC with 1.194 purity (A620/A280) was obtained via autolysis after 3 h of incubation at 37 °C without using an extraction salt solution or a successive freezing-thawing process. In addition to temperature, the initial concentration of bicarbonate in growth medium and the concentration of wet biomass are important parameters that influence the extraction yield of PC by autolysis.

Potential of Bacterial Strains Isolated from Coastal Water for Wastewater Treatment and as Aqua-Feed Additives.

Bacteria have various and sustained effects on humans in various fields: molecular biology, biomedical science, environmental/food industry, etc. This study was conducted to evaluate the wastewater treatment capacity and feed-additive fish-growth effect of four strains of bacteria: Pseudoalteromonas mariniglutinosa, Psychrobacter celer, Bacillus albus, and Bacillus safensis. In a wastewater degradation experiment, (i) nitrate-N and nitrite-N were removed within 1 h in all of the 4 bacterial strains; (ii) the removal rates of TAN and TN were higher in all of the strains relative to the B. subtilis. In a feed-additive experiment (5% Kg-1), (i) the growth of fish was higher in all of the 4 bacterial strains with the B. subtilis relative to the commercial feed; (ii) there was no significant growth difference for B. albus and B. safensis relative to the B. subtilis, but growth was higher in P. mariniglutinosa and P. celer. The results indicated that the 4 bacterial strains can be effectively utilized for biological wastewater treatment processes and as aqua-feed.

Non-toxic nano approach for wastewater treatment using Chlorella vulgaris exopolysaccharides immobilized in iron-magnetic nanoparticles.

The current study, novel magnetic nano-composite particles (Fe3O4@EPS) were successfully synthesized via the co-precipitation of iron (III) chloride and iron (II) sulfate (Fe3O4 nanoparticles) with exopolysaccharides (EPS) derived from the microalga Chlorella vulgaris. The physico-chemical nature of the Fe3O4@EPS was investigated in depth. Transmission electron microscopy (TEM) results estimated the core-shell nature of Fe3O4@EPS aggregated inside the indistinctly layered EPS matrix to be 10-20 nm in size. Scanning electron microscopy-based energy dispersive spectral analysis indicated that elemental Fe was successfully loaded on to the EPS polymeric ion-exchanger at a rate of 63.3% by weight. FT-IR results demonstrated that Fe3O4 nanoparticles were successfully modified by the functional groups present in EPS. Fe3O4@EPS showed a highly magnetic nature at 5.0 emu/g. The XPS survey spectrum, which showed two major peaks at 724.1 and 710.2 eV revealed the elemental composition and electronic structure of Fe3O4 nanoparticles and Fe3O4@EPS. Furthermore, nutrient removal from wastewater was studied. Under optimum conditions (3.5 g/L of Fe3O4@EPS, pH 7.0 and 13 h of incubation) 91% of PO43- and 85% of NH4+were effectively eliminated. These findings demonstrate the potential of Fe3O4@EPS for removing PO43- and NH4+ in wastewater treatment plants.

Growth medium sterilization using decomposition of peracetic acid for more cost-efficient production of omega-3 fatty acids by Aurantiochytrium.

Aurantiochytrium can produce significant amounts of omega-3 fatty acids, specifically docosahexaenoic acid and docosapentaenoic acid. Use of a glucose-based medium for heterotrophic growth is needed to achieve a high growth rate and production of abundant lipids. However, heat sterilization for reliable cultivation is not appropriate to heat-sensitive materials and causes a conversion of glucose via browning (Maillard) reactions. Thus, the present study investigated the use of a direct degradation of Peracetic acid (PAA) for omega-3 production by Aurantiochytrium. Polymer-based bioreactor and glucose-containing media were chemically co-sterilized by 0.04% PAA and neutralized through a reaction with ferric ion (III) in HEPES buffer. Mono-cultivation was achieved without the need for washing steps and filtration, thereby avoiding the heat-induced degradation and dehydration of glucose. Use of chemically sterilized and neutralized medium, rather than heat-sterilized medium, led to a twofold faster growth rate and greater productivity of omega-3 fatty acids.

Complementation of a mutation in CpSRP43 causing partial truncation of light-harvesting chlorophyll antenna in Chlorella vulgaris.

Photosynthesis of microalgae enables conversion of light energy into chemical energy to produce biomass and biomaterials. However, the efficiency of this process must be enhanced, and truncation of light-harvesting complex (LHC) has been suggested to improve photosynthetic efficiency. We reported an EMS-induced mutant (E5) showing partially reduced LHC in Chlorella vulgaris. We determined the mutation by sequencing the whole genome of WT and E5. Augustus gene prediction was used for determining CDS, and non-synonymous changes in E5 were screened. Among these, we found a point mutation (T to A) in a gene homologous to chloroplast signal recognition particle 43 kDa (CpSRP43). The point mutation changed the 102nd valine to glutamic acid (V102E) located in the first chromodomain. Phylogenetic analyses of CpSRP43 revealed that this amino acid was valine or isoleucine in microalgae and plants, suggesting important functions. Transformation of E5 with WT CpSRP43 showed varying degrees of complementation, which was demonstrated by partial recovery of the LHCII proteins to the WT level, and partially restored photosynthetic pigments, photosynthetic ETR, NPQ, and growth, indicating that the V102E mutation was responsible for the reduced LHC in E5.

A Simple, Cost-Efficient Method to Separate Microalgal Lipids from Wet Biomass Using Surface Energy-Modified Membranes.

For the efficient separation of lipid extracted from microalgae cells, a novel membrane was devised by introducing a functional polymer coating onto a membrane surface by means of an initiated chemical vapor deposition (iCVD) process. To this end, a steel-use-stainless (SUS) membrane was modified in a way that its surface energy was systemically modified. The surface modification by conformal coating of functional polymer film allowed for selective separation of oil-water mixture, by harnessing the tuned interfacial energy between each liquid phase and the membrane surface. The surface-modified membrane, when used with chloroform-based solvent, exhibited superb permeate flux, breakthrough pressure, and also separation yield: it allowed separation of 95.5 ± 1.2% of converted lipid (FAME) in the chloroform phase from the water/MeOH phase with microalgal debris. This result clearly supported that the membrane-based lipid separation is indeed facilitated by way of membrane being functionalized, enabling us to simplify the whole downstream process of microalgae-derived biodiesel production.

Use of a triiodide resin for isolation of axenic cultures of microalgal Nannochloropsis gaditana.

Triiodide resin (TR) was used to generate axenic cultures of microalgae by employing the antibacterial capability of triiodide. A Nannochloropsis gaditana culture contaminated with bacteria was passed through a column filled with TR using the gravity flow. Based on analyses of flow cytometry and vital staining using a fluorescent dye SYTOX Green, three cycles of TR treatments remarkably reduced the number of viable bacteria but had little effects on the microalgae. This novel approach is a simple, rapid, and cost-effective method that can be used to isolate axenic cultures of microalgae.

Optimization of variables affecting the direct transesterification of wet biomass from Nannochloropsis oceanica using ionic liquid as a co-solvent.

Ionic liquids have many applications, one of which entails their utilization as powerful solvents. In the present study, various experimental conditions of ionic liquid-mediated direct transesterification were investigated in terms of lipid-extracting ionic liquids, catalyst, reaction time, reaction temperature and volume of methanol to achieve effective FAME conversion with wet microalgal feedstock, Nannochloropsis oceanica. With ionic liquid, [Bmim][CF3SO3], highest fatty acid methyl ester (FAME) yield was shown. Among many experimental parameters, the two most critical factors to enhance FAME conversion were characteristic of ionic liquids and volume of methanol. Optimized ionic liquid-mediated direct transesterification of wet N. oceanica, compared with a control experiment using chloroform and methanol, increased the FAME conversion yield by 11-fold.

A simple method for decomposition of peracetic acid in a microalgal cultivation system.

A cost-efficient process devoid of several washing steps was developed, which is related to direct cultivation following the decomposition of the sterilizer. Peracetic acid (PAA) is known to be an efficient antimicrobial agent due to its high oxidizing potential. Sterilization by 2 mM PAA demands at least 1 h incubation time for an effective disinfection. Direct degradation of PAA was demonstrated by utilizing components in conventional algal medium. Consequently, ferric ion and pH buffer (HEPES) showed a synergetic effect for the decomposition of PAA within 6 h. On the contrary, NaNO3, one of the main components in algal media, inhibits the decomposition of PAA. The improved growth of Chlorella vulgaris and Synechocystis PCC6803 was observed in the prepared BG11 by decomposition of PAA. This process involving sterilization and decomposition of PAA should help cost-efficient management of photobioreactors in a large scale for the production of value-added products and biofuels from microalgal biomass.

Cultivation of Chlorella protothecoides in anaerobically treated brewery wastewater for cost-effective biodiesel production.

The use of wastewater has been investigated to overcome the economic challenge involved with a production of microalgae-based biodiesel. In this study, to achieve economical biodiesel production along with effective wastewater treatment at the same time, anaerobically treated brewery wastewater (ABWW) was utilized as a low-cost nutrient source, in the cultivation of Chlorella protothecoides. About 96 and 90 % of total nitrogen and phosphorus in ABWW were removed, respectively, while C. protothecoides was accumulating 1.88 g L(-1) of biomass. The C. protothecoides grown in ABWW showed increases in cell size and cell aggregation, resulting in a near 80 % enhanced harvesting efficiency within 20 min, as compared with only 4 % in BG-11. In addition, the total fatty acid content of the C. protothecoides grown in ABWW increased by 1.84-fold (35.94 ± 1.54 % of its dry cell weight), relative to that of BG-11.

Utilization of seawater for cost-effective cultivation and harvesting of Scenedesmus obliquus.

Microalgae hold great promise as a source of biofuels and biochemicals. The main obstacles to their industrial application are the high cultivation and downstream costs related to media and harvesting. In the work, we explored the multiple potentials of seawater to address key issues relating to the cultivation of Scenedesmus obliquus. Seawater can sufficiently replace some of the key elements in BG11 medium such as MgSO4, CaCl2, and NaCO3, and its use can significantly reduce the quantity of water required for the preparation of culture media. Among our results, the total chlorophyll content in cells grown in modified BG11 using 10 % (v/v) seawater was increased 1.47-fold without sacrificing biomass or lipid production. More than 70 % of the total algal biomass was auto-flocculated within one hour when cells were grown in seawater-supplemented media, which compares very favorably with a yield of only 3 % from cells grown in BG11.

Use of extracts from oyster shell and soil for cultivation of Spirulina maxima.

Calcium ion and trace metals play important roles in various metabolisms of photosynthetic organisms. In this study, simple methods were developed to extract calcium ion and micronutrients from oyster shell and common soil, and the prepared extracts were tested as a replacement of the corresponding chemicals that are essential for growth of microalgae. The oyster shell and soil were treated with 0.1 M sodium hydroxide or with 10 % hydrogen peroxide, respectively. The potential application of these natural sources to cultivation was investigated with Spirulina maxima. When compared to standard Zarrouk medium, the Spirulina maxima cultivated in a modified Zarrouk media with elements from oyster shell and soil extract exhibited increases in biomass, chlorophyll, and phycocyanin by 17, 16, and 64 %, respectively. These results indicate that the extracts of oyster shell and soil provide sufficient amounts of calcium and trace metals for successful cultivation of Spirulina maxima.

Effects of molten-salt/ionic-liquid mixture on extraction of docosahexaenoic acid (DHA)-rich lipids from Aurantiochytrium sp. KRS101.

In this study, lipid extraction from Aurantiochytrium sp. was performed using a molten-salt/ionic-liquid mixture. The total fatty acid content of Aurantiochytrium sp. was 478.8 mg/g cell, from which 145 mg/g cell (30.3% of total fatty acids) of docosahexaenoic acid (DHA) was obtained. FeCl3·6H2O showed a high lipid extraction yield (207.9 mg/g cell), when compared with that of [Emim]OAc, which was only 118.1 mg/g cell; notably however, when FeCl3·6H2O was mixed with [Emim]OAc (5:1, w/w), the yield was increased to 478.6 mg/g cell. When lipid was extracted by the FeCl3·6H2O/[Emim]OAc mixture at a 5:1 (w/w) blending ratio under 90 °C, 30 min reaction conditions, the fatty acid content of the extracted lipid was a high purity 997.7 mg/g lipid, with most of the DHA having been extracted (30.2% of total fatty acids). Overall, lipid extraction from Aurantiochytrium sp. was enhanced by the synergistic effects of the molten-salt/ionic-liquid mixture with different ions.

Acid-catalyzed hot-water extraction of docosahexaenoic acid (DHA)-rich lipids from Aurantiochytrium sp. KRS101.

In this study, acid-catalyzed hot-water extraction of docosahexaenoic acid (DHA)-rich lipids from Aurantiochytrium sp. was performed, and its yield-enhancing effects were investigated. The total fatty acid content of the Aurantiochytrium sp. was 482.5mg/g cell, of which 141.7mg/g cell (29.4% of total fatty acids) was DHA. The lipid-extraction yield by acid-catalyzed hot-water treatment was compared with those by organic solvents. Among the various acid-catalyzed hot-water treatment conditions, the most optimal were 1.00% H2SO4 concentration, 100°C, 30min, under which the lipid-extraction yield was 472.4mg/g cell, and most of the DHA was extracted (29.2% of total fatty acids). Acid-catalyzed hot-water extraction treatment markedly improved the lipid-extraction yield of Aurantiochytrium sp.

Effect of monochromatic illumination on lipid accumulation of Nannochloropsis gaditana under continuous cultivation.

Although nitrogen starvation is frequently used to increase lipid contents in microalgae, it has a negative effect on cellular growth. Since light supply is essential for photosynthetic organisms, the effects of cultivation under monochromatic illumination on the growth and lipid contents of Nannochloropsis gaditana were assessed. Continuous cultivation under blue and red light conditions improved the productivity and physical properties for biodiesel from this microalga. FAME yield was twofold higher under red light than under normal white light (21.12% vs 11.35%), with no significant difference in growth rates. Blue and red light increased photosynthetic oxygen evolution, carbon fixation and nutrient uptake. In total, more significant physiological changes were observed under red than under blue light. These results show that red light illumination may be useful for enhancing lipid production by N. gaditana, with the increased photosynthetic reducing equivalents induced by red light which could be deposited as lipids and carbohydrates.

Direct approach for bioprocess optimization in a continuous flat-bed photobioreactor system.

Application of photosynthetic micro-organisms, such as cyanobacteria and green algae, for the carbon neutral energy production raises the need for cost-efficient photobiological processes. Optimization of these processes requires permanent control of many independent and mutably dependent parameters, for which a continuous cultivation approach has significant advantages. As central factors like the cell density can be kept constant by turbidostatic control, light intensity and iron content with its strong impact on productivity can be optimized. Both are key parameters due to their strong dependence on photosynthetic activity. Here we introduce an engineered low-cost 5 L flat-plate photobioreactor in combination with a simple and efficient optimization procedure for continuous photo-cultivation of microalgae. Based on direct determination of the growth rate at constant cell densities and the continuous measurement of O2 evolution, stress conditions and their effect on the photosynthetic productivity can be directly observed.