Secretomics: a biochemical footprinting tool for developing microalgal cultivation strategies.

Microalgae offer a promising source of biofuel and a wide array of high-value biomolecules. Large-scale cultivation of microalgae at low density poses a significant challenge in terms of water management. High-density microalgae cultivation, however, can be challenging due to biochemical changes associated with growth dynamics. Therefore, there is a need for a biomarker that can predict the optimum density for high biomass cultivation. A locally isolated microalga Cyanobacterium aponinum CCC734 was grown with optimized nitrogen and phosphorus in the ratio of 12:1 for sustained high biomass productivity. To understand density-associated bottlenecks secretome dynamics were monitored at biomass densities from 0.6 ± 0.1 to 7 ± 0.1 g/L (2 to 22 OD) in batch mode. Liquid chromatography coupled with mass spectrometry identified 880 exometabolites in the supernatant of C. aponinum CCC734. The PCA analysis showed similarity between exometabolite profiles at low (4 and 8 OD) and mid (12 and 16 OD), whereas distinctly separate at high biomass concentrations (20 and 22 OD). Ten exometabolites were selected based on their role in influencing growth and are specifically present at low, mid, and high biomass concentrations. Taking cues from secretome dynamics, 5.0 ± 0.5 g/L biomass concentration (16 OD) was optimal for C. aponinum CCC734 cultivation. Further validation was performed with a semi-turbidostat mode of cultivation for 29 days with a volumetric productivity of 1.0 ± 0.2 g/L/day. The secretomes-based footprinting tool is the first comprehensive growth study of exometabolite at the molecular level at variable biomass densities. This tool may be utilized in analyzing and directing microalgal cultivation strategies and reduction in overall operating costs.

Mineral phosphorus drives glacier algal blooms on the Greenland Ice Sheet.

Melting of the Greenland Ice Sheet is a leading cause of land-ice mass loss and cryosphere-attributed sea level rise. Blooms of pigmented glacier ice algae lower ice albedo and accelerate surface melting in the ice sheet's southwest sector. Although glacier ice algae cause up to 13% of the surface melting in this region, the controls on bloom development remain poorly understood. Here we show a direct link between mineral phosphorus in surface ice and glacier ice algae biomass through the quantification of solid and fluid phase phosphorus reservoirs in surface habitats across the southwest ablation zone of the ice sheet. We demonstrate that nutrients from mineral dust likely drive glacier ice algal growth, and thereby identify mineral dust as a secondary control on ice sheet melting.

Laser Capture Microdissection-Liquid Vortex Capture Mass Spectrometry Metabolic Profiling of Single Onion Epidermis and Microalgae Cells.

Laser capture microdissection is a valuable technique in individually isolating single cells whether in tissue networks or deposited from a cell suspension. New developments have enabled coupling of laser capture microdissection with mass spectrometry via liquid vortex capture sampling probe. This enables online metabolic profiling of sectioned cells. Here, we describe the protocol used to deposit, isolate, and individually chemically characterize single Allium cepa and Chlamydomonas reinhardtii cells by laser capture microdissection-liquid vortex capture mass spectrometry.

Auto-flocculation through cultivation of Chlorella vulgaris in seafood wastewater discharge: Influence of culture conditions on microalgae growth and nutrient removal.

Nowadays, the pretreatment of wastewater prior to discharge is very important in various industries as the wastewater without any treatment contains high organic pollution loads that would pollute the receiving waterbody and potentially cause eutrophication and oxygen depletion to aquatic life. The reuse of seafood wastewater discharge in microalgae cultivation offers beneficial purposes such as reduced processing cost for wastewater treatment, replenishing ground water basin as well as financial savings for microalgae cultivation. In this paper, the cultivation of Chlorella vulgaris with an initial concentration of 0.01 ± 0.001 g⋅L-1 using seafood sewage discharge under sunlight and fluorescent illumination was investigated in laboratory-scale without adjusting mineral nutrients and pH. The ability of nutrient removal under different lighting conditions, the metabolism of C. vulgaris and new medium as well as the occurrence of auto-flocculation of microalgae biomass were evaluated for 14 days. The results showed that different illumination sources did not influence the microalgae growth, chemical oxygen demand (COD) and biochemical oxygen demand (BOD) significantly. However, the total nitrogen (total-N) and total phosphorus (total-P) contents of microalgae were sensitive to the illumination mode. The amount of COD, BOD, total-N and total-P were decreased by 88%, 81%, 95%, and 83% under sunlight mode and 81%, 74%, 79%, and 72% under fluorescent illumination, respectively. Furthermore, microalgae were auto-flocculated at the final days of cultivation with maximum biomass concentration of 0.49 ± 0.01 g⋅L-1, and the pH value had increased to pH 9.8 ± 0.1 under sunlight illumination.

Development of a whole-cell-based screening method for a carotenoid assay using aerial microalgae.

Non-destructive approaches based on the application of optical spectroscopy are important for monitoring carotenoid accumulation in a whole cell cultured under various conditions. A simple and rapid assay utilizing aerial microalgae helps to identify stress conditions that can efficiently enhance the carotenogenesis in photosynthetic organisms. The spectra of cell suspensions were characterized in the aerial microalga Coelastrella sp. KGU-Y002, which are unicellular and undifferentiated. Total carotenoid contents could be successfully estimated on the basis of the absorbance values of the cell suspensions and calibration data analyzed by HPLC (high-performance liquid chromatography). A novel screening method, the so-called "whole-cell-based screening method" for carotenoid assays (WCA), was developed based on this procedure. It was possible to investigate the effects of various stresses on carotenoid accumulation in the aerial microalga by adapting this bioassay to a 96-well microtiter plate. When bioactive compounds were screened from our library of plant extracts using this method, an active compound was identified from the plant extract.

Advances in high frequency ultrasound separation of particulates from biomass.

In recent years the use of high frequency ultrasound standing waves (megasonics) for droplet or cell separation from biomass has emerged beyond the microfluidics scale into the litre to industrial scale applications. The principle for this separation technology relies on the differential positioning of individual droplets or particles across an ultrasonic standing wave field within the reactor and subsequent biomass material predisposition for separation via rapid droplet agglomeration or coalescence into larger entities. Large scale transducers have been characterised with sonochemiluminescence and hydrophones to enable better reactor designs. High frequency enhanced separation technology has been demonstrated at industrial scale for oil recovery in the palm oil industry and at litre scale to assist olive oil, coconut oil and milk fat separation. Other applications include algal cell dewatering and milk fat globule fractionation. Frequency selection depends on the material properties and structure in the biomass mixture. Higher frequencies (1 and 2MHz) have proven preferable for better separation of materials with smaller sized droplets such as milk fat globules. For palm oil and olive oil, separation has been demonstrated within the 400-600kHz region, which has high radical production, without detectable impact on product quality.

Boosting TAG Accumulation with Improved Biodiesel Production from Novel Oleaginous Microalgae Scenedesmus sp. IITRIND2 Utilizing Waste Sugarcane Bagasse Aqueous Extract (SBAE).

This investigation utilized sugarcane bagasse aqueous extract (SBAE), a nontoxic, cost-effective medium to boost triacylglycerol (TAG) accumulation in novel fresh water microalgal isolate Scenedesmus sp. IITRIND2. Maximum lipid productivity of 112 ± 5.2 mg/L/day was recorded in microalgae grown in SBAE compared to modified BBM (26 ± 3 %). Carotenoid to chlorophyll ratio was 12.5 ± 2 % higher than in photoautotrophic control, indicating an increase in photosystem II activity, thereby increasing growth rate. Fatty acid methyl ester (FAME) profile revealed presence of C14:0 (2.29 %), C16:0 (15.99 %), C16:2 (4.05 %), C18:0 (3.41 %), C18:1 (41.55 %), C18:2 (12.41), and C20:0 (1.21 %) as the major fatty acids. Cetane number (64.03), cold filter plugging property (-1.05 °C), and oxidative stability (12.03 h) indicated quality biodiesel abiding by ASTM D6751 and EN 14214 fuel standards. Results consolidate the candidature of novel freshwater microalgal isolate Scenedesmus sp. IITRIND2 cultivated in SBAE, aqueous extract made from copious, agricultural waste sugarcane bagasse to increase the lipid productivity, and could further be utilized for cost-effective biodiesel production.

Triacylglycerol Accumulation in Photosynthetic Cells in Plants and Algae.

Plant and algal oils are some of the most energy-dense renewable compounds provided by nature. Triacylglycerols (TAGs) are the major constituent of plant oils, which can be converted into fatty acid methyl esters commonly known as biodiesel. As one of the most efficient producers of TAGs, photosynthetic microalgae have attracted substantial interest for renewable fuel production. Currently, the big challenge of microalgae based TAGs for biofuels is their high cost compared to fossil fuels. A conundrum is that microalgae accumulate large amounts of TAGs only during stress conditions such as nutrient deprivation and temperature stress, which inevitably will inhibit growth. Thus, a better understanding of why and how microalgae induce TAG biosynthesis under stress conditions would allow the development of engineered microalgae with increased TAG production during conditions optimal for growth. Land plants also synthesize TAGs during stresses and we will compare new findings on environmental stress-induced TAG accumulation in plants and microalgae especially in the well-characterized model alga Chlamydomonas reinhardtii and a biotechnologically relevant genus Nannochloropsis.

Microalgal growth with intracellular phosphorus for achieving high biomass growth rate and high lipid/triacylglycerol content simultaneously.

Nutrient deprivation is a commonly-used trigger for microalgal lipid accumulation, but its adverse impact on microalgal growth seems to be inevitable. In this study, Scenedesmus sp. LX1 was found to show similar physiological and biochemical variation under oligotrophic and eutrophic conditions during growth with intracellular phosphorus. Under both conditions microalgal chlorophyll content and photosynthesis activity was stable during this growth process, leading to significant increase of single cell weight and size. Therefore, while algal density growth rate dropped significantly to below 1.0 × 10(5)cells mL(-1) d(-1) under oligotrophic condition, the biomass dry weight growth rate still maintained about 40 mg L(-1) d(-1). Meanwhile, the lipid content in biomass and triacylglycerols (TAGs) content in lipids increased significantly to about 35% and 65%, respectively. Thus, high biomass growth rate and high lipid/TAG content were achieved simultaneously at the late growth phase with intracellular phosphorus. Besides, microalgal biomass produced was rich in carbohydrate with low protein content.

The characteristics of TAG and EPA accumulation in Nannochloropsis oceanica IMET1 under different nitrogen supply regimes.

The strategy of nitrogen limitation has been widely applied to enhance lipid production in microalgae. The changes of cellular composition, and the characteristics of triacylglycerol (TAG) and eicosapentaenoic acid (EPA) accumulation in Nannochloropsis oceanica IMET1 were investigated. The results revealed that after nitrogen limitation TAG rather than carbohydrate was the dominant carbon sink in N. oceanica IMET1. Different nitrogen supplementation strategies were applied in order to achieve high TAG and EPA productivity, respectively. Limited nitrogen was supplied to improve TAG production, and a maximum productivity of 29.44 mg L(-1) d(-1) was obtained, which was a 6.74-fold increase compared to nitrogen-depleted cultivation. The highest EPA productivity of 7.66 mg L(-1) d(-1) was achieved under nitrogen-replete cultivation, which is different from the condition for TAG maximum productivity because the EPA is in glycolipids and phospholipids mainly. The fatty acid composition analysis identified the source of acyl group in TAG accumulation.

Efficacy of binary combinations of botanical pesticides for rotifer elimination in microalgal cultivation.

Binary interactions of celangulin, matrine and toosendanin against the rotifer Brachionus plicatilis were studied. Types of interactions (antagonism, synergism and addition) were dependent on the biocides themselves and their ratios in combinations. Mixtures of matrine/toosendanin mainly produced addition owing to their similar modes of action aiming at the nervous system. Combinations of celangulin mixed with matrine or toosendanin at 1:9 exhibited synergism, which is attributed to the interference of matrine or toosendanin with the detoxification enzymes of celangulin. Both the synergistic combinations were inappropriate for rotifer extermination in Isochrysis sp. cultivation owing to the high phytotoxicity resulting from the absence of cell walls. However, the celangulin/toosendanin (1:9) mixture decreased rotifer reproduction without damaging cells of Chlorella and Nannochloropsis sp. Application of frequent, low doses of celangulin/toosendanin (1:9) mixture also reduced the dosage of biocides, thereby reducing the cost of exterminating rotifers, and indicating a considerable practical application in microalgal cultivation.

Application of a prototype-scale Twin-Layer photobioreactor for effective N and P removal from different process stages of municipal wastewater by immobilized microalgae.

In the view of limited phosphorous resources and tightened discharge regulations, the recovery of phosphate and nitrate from wastewater is of great interest. Here, the integration of microalgae into wastewater treatment processes is a promising approach. A prototype-scale Twin-Layer photobioreactor immobilizing the green alga Halochlorella rubescens on vertical sheet-like surfaces was constructed and operated using primary and secondary municipal wastewater. The process was not impaired by suspended solids, bacteria or loss of algal biomass by leaching. The average areal microalgal growth was 6.3 gm(-2) d(-1). After treatment, P and N concentrations in the effluents could efficiently be reduced by 70-99%, depending on element and type of wastewater. Mean effluent values of ⩽ 1.0mg L(-1)P and 1.3 mg L(-1)N met the legal discharge limits of the European Water Framework Directive and show a potential to comply with upcoming, more stringent legislation.

Relationship between starch and lipid accumulation induced by nutrient depletion and replenishment in the microalga Parachlorella kessleri.

Photosynthetic carbon partitioning into starch and neutral lipids, as well as the influence of nutrient depletion and replenishment on growth, pigments and storage compounds, were studied in the microalga, Parachlorella kessleri. Starch was utilized as a primary carbon and energy storage compound, but nutrient depletion drove the microalgae to channel fixed carbon into lipids as secondary storage compounds. Nutrient depletion inhibited both cellular division and growth and caused degradation of chlorophyll. Starch content decreased from an initial value of 25, to around 10% of dry weight (DW), while storage lipids increased from almost 0 to about 29% of DW. After transfer of cells into replenished mineral medium, growth, reproductive processes and chlorophyll content recovered within 2 days, while the content of both starch and lipids decreased markedly to 3 or less % of DW; this suggested that they were being used as a source of energy and carbon.

Selection and evaluation of CO2 tolerant indigenous microalga Scenedesmus dimorphus for unsaturated fatty acid rich lipid production under different culture conditions.

Five indigenous microalgal strains of Scenedesmus, Chlorococcum, Coelastrum, and Ankistrodesmus genera, isolated from Indian fresh water habitats, were studied for carbon-dioxide tolerance and its effect on growth, lipid and fatty acid profile. Scenedesmus dimorphus strain showed maximum growth (1.5 g/L) and lipid content (17.83% w/w) under CO2 supplementation, hence selected for detailed evaluation. The selected strain was alkaline adapted but tolerated (i) wide range of pH (5-11); (ii) elevated salinity levels (up to 100 mM, NaCl) with low biomass yields and increased carotenoids (19.34 mg/g biomass); (iii) elevated CO2 levels up to 15% v/v with enhancement in specific growth rate (0.137 d(-1)), biomass yield (1.57 g/L), lipid content (19.6% w/w) and CO2 biofixation rate (0.174 g L(-1) d(-1)). Unsaturated fatty acid content (alpha linolenic acid) increased with CO2 supplementation in the strain.

Nutrient sequestration, biomass production by microalgae and phytoremediation of sewage water.

The present work was aimed at analysing the role of inoculated microalgae in nutrient dynamics, bioremediation and biomass production of sewage water. Preliminary microscopic analyses of sewage water revealed the presence of different algal groups, with predominance of Cyanophyta. Among the inoculated strains, Calothrix showed highest dry cell weight (916.67 mg L(-1)), chlorophyll and carotenoid content in tap water + sewage water (1:1) treatment. Significant removal of NO3-N ranging from 57-78% and PO4-P (44-91%) was recorded in microalgae inoculated tap water + sewage water. The total dissolved solids and electrical conductivity of tap water + sewage water after incubation with Calothrix sp. decreased by 28.5 and 28.0%, accompanied by an increase in dissolved oxygen from 4.4 to 6.4 mg L(-1) on the 20th day. Our investigation revealed the robustness of Calothrix sp. in sequestering nutrients (N and P), improving water quality and proliferating in sewage water.

Application of mid-infrared chemical imaging and multivariate chemometrics analyses to characterise a population of microalgae cells.

A suite of multivariate chemometrics methods was applied to a mid-infrared imaging dataset of a eustigmatophyte, marine Nannochloropsis sp. microalgae strain. This includes the improved leader-follower cluster analysis (iLFCA) to interrogate spectra in an unsupervised fashion, a resonant Mie optical scatter correction algorithm (RMieS-EMSC) that improves data linearity, the band-target entropy minimization (BTEM) self-modeling curve resolution for recovering component spectra, and a multi-linear regression (MLR) for estimating relative concentrations and plotting chemical maps of component spectra. A novel Alpha-Stable probability calculation for microalgae cellular lipid-to-protein ratio Λi is introduced for estimating population characteristics.

Laser desorption/ionization mediated by bionanostructures from microalgae.

RATIONALE: Organic matrices are the state-of-the-art ionization mediators in Laser Desorption/Ionization Mass Spectrometry (LDI-MS). Despite improvements in understanding matrix chemistry, interfering matrix-related signals complicate the analysis. Surface-assisted LDI techniques like desorption/ionization on silicon (DIOS) or nanostructure initiator mass spectrometry (NIMS) provide promising alternatives but rely often on elaborate materials. METHODS: We introduce nanopatterned biomineralized cell walls of microalgae as easily accessible biological surfaces that support the ionization of embedded molecules in LDI-MS. Microalgae cell walls were cleaned through oxidation and washing before pipetting on a stainless-steel matrix-assisted laser desorption/ionization (MALDI) target. Added molecules were efficiently ionized in positive and negative ionization mode in common MALDI sources. The method was rigorously validated by comparison with established MALDI experiments. RESULTS: Ionization of PEG600, D-sphingosine and raffinose was successfully mediated by nanostructured cell wall preparations from two different microalgae. Without any change in protocol, steric acid could be detected in the negative ionization mode. Ionization is also supported by commercially available celite, a material containing mineralized diatom cell walls. Characteristic ingredients of fresh coffee were detected in LDI-MS after pipetting it on celite without further sample preparation. Caffeine and saccharose were detected in positive and characteristic fatty acids in negative ionization mode. Detection limits were comparable to established MALDI experiments. CONCLUSIONS: Bionanostructure-enhanced ionization allows the analysis of a diverse selection of analytes including polymers, sugars, amino alcohols, and organic acids without interfering matrix signals. We also show that celite, a commercially available porous material containing mineralized algal bionanostructures, supports LDI-MS.

Algal diversity in flowing waters at an acidic mine drainage "barrens" in central Pennsylvania, USA.

Microscopic investigations were undertaken to decipher the diversity in the lotic algal communities from acidic waters (pH 2.4-3.2) flowing overland in sheets and channels at an acid mine drainage (AMD) barrens near Kylertown, PA, USA. Microscopic observations, supplemented with taxonomic keys, aided in identification of the dominant algae, and measurement of carbon from adjacent soils was undertaken. The unicellular protist Euglena sp. was most abundant in slower flowing waters (i.e., pool near point of emergence and surficial flow sheets), while Ulothrix sp. was most abundant in faster flowing water from the central stream channel. A diverse range of unicellular microalgae such as Chlorella, Cylindrocystis, Botryococcus, and Navicula and several filamentous forms identified as Microspora, Cladophora, and Binuclearia were also recorded. The observed high algal diversity may be related to the long duration of AMD flow at this site which has led to the development of adapted algal communities. The comparatively higher carbon content in soil materials adjacent to slower flowing water sampling locations provides evidence for the important role of algae as primary producers in this extreme environment.