Abiotic and Biotic Damage of Microalgae Generate Different Volatile Organic Compounds (VOCs) for Early Diagnosis of Algal Cultures for Biofuel Production

Open microalgal ponds used in industrial biomass production are susceptible to a number of biotic and abiotic environmental stressors (e.g., grazers, pathogens, pH, temperature, etc.) resulting in pond crashes with high economic costs. Identification of signature chemicals to aid in rapid, non-invasive, and accurate identification of the stressors would facilitate targeted and effective treatment to save the algal crop from a catastrophic crash. Specifically, we were interested in identifying volatile organic compounds (VOCs) that can be used to as an early diagnostic for algal crop damage. Cultures of Microchloropsis gaditana were subjected to two forms of algal crop damage: (1) active grazing by the marine rotifer, Brachionus plicatilis, or (2) repeated freeze–thaw cycles. VOCs emitted above the headspace of these algal cultures were collected using fieldable solid phase microextraction (SPME) fibers. An untargeted analysis and identification of VOCs was conducted using gas chromatography-mass spectrometry (GC-MS). Diagnostic VOCs unique to each algal crop damage mechanism were identified. Active rotifer grazing of M. gaditana was characterized by the appearance of carotenoid degradation products, including β-cyclocitral and various alkenes. Freeze–thaw algae produced a different set of VOCs, including palmitoleic acid. Both rotifer grazing and freeze–thawed algae produced β-ionone as a VOC, possibly suggesting a common stress-induced cellular mechanism. Importantly, these identified VOCs were all absent from healthy algal cultures of M. gaditana. Early detection of biotic or abiotic environmental stressors will facilitate early diagnosis and application of targeted treatments to prevent algal pond crashes. Thus, our work further supports the use of VOCs for monitoring the health of algal ponds to ultimately enhance algal crop yields for production of biofuel.

Approaches and involved principles to control pH/pCO2 stability in algal cultures

Experimental cultures of both microalgae and macroalgae are commonly carried out by phycologists or environmental biologists to look into morphological, physiological, and molecular responses to aquatic environmental changes. However, the species of inorganic carbon in algae cultures is often altered by algal photosynthetic CO2 removal and/or bicarbonate utilization. The pH changes associated with altered carbonate chemistry in cultures impact physiological processes in microalgae and macroalgae even at their exponential growth phases, since extra energy is required to sustain intracellular acid–base homeostasis. Usually, pH increases during light period due to inorganic carbon uptake and utilization for photosynthesis and decreases during dark period because of respiratory CO2 release. Therefore, to obtain relevant data aimed for physiological and/or molecular responses of algae to changed levels of environmental factors, stability of pH/pCO2 in the cultures should be considered and controlled to rule out impacts of carbonate chemistry and pH changes. In this work, principles involved in changing pH processes in algal cultures are mechanistically analyzed and several approaches to control pH and pCO2 are introduced. In order to sustain stability of pH/pCO2, the principles underline the following key points: (1) maintaining the rate of photosynthetic C removal less than or equal to the rate of CO2 dissolution into the cultures which are aerated; or (2) sustaining dilute cultures with very low cell density without aeration, so that photosynthetic C removal is small enough not to cause significant pH/pCO2 changes; or (3) stabilizing the changes in micro-environments surrounding the cells or thallus. To maintain pH drift < 1% in growing typical unicellular microalgae, the recommended cell concentration ranges from 50 × 103 to 200 × 103 mL−1 with aeration (air replacement rate of ca 500–1000 mL L−1 min−1) in semi-continuous cultures of < 1 L, and it ranges from 100 to 5000 cells mL−1 for diatoms and from 100 to 100 × 103 cells mL−1 for coccolithophores in dilute cultures without aeration, respectively. For macroalgae, maintaining the thalli in flowing through- system or in semi-continuous cultures (continuously control algal biomass density) is recommended.

Identification of effector metabolites using exometabolite profiling of diverse microalgae

Dissolved metabolites mediate algal interactions in aquatic ecosystems, but microalgal exometabolomes remain understudied. We conducted an untargeted metabolomic analysis of non-polar exometabolites exuded from four phylogenetically and ecologically diverse eukaryotic microalgal strains grown in the laboratory: freshwater Chlamydomonas reinhardtii, brackish Desmodesmus sp., marine Phaeodactylum tricornutum, and marine Microchloropsis salina, to identify released metabolites based on relative enrichment in the exometabolomes compared to cell pellet metabolomes. Exudates from the different taxa were distinct, but we did not observe clear phylogenetic patterns. We used feature based molecular networking to explore the identities of these metabolites, revealing several distinct di- and tripeptides secreted by each of the algae, lumichrome, a compound that is known to be involved in plant growth and bacterial quorum sensing, and novel prostaglandin-like compounds. We further investigated the impacts of exogenous additions of eight compounds selected based on exometabolome enrichment on algal growth. Of the these, five (lumichrome, 5’-S-methyl-5'-thioadenosine, 17-phenyl trinor prostaglandin A2, dodecanedioic acid, and aleuritic acid) impacted growth in at least one of the algal cultures. Two of these (dodecanedioic acid and aleuritic acid) produced contrasting results, increasing growth in some algae and decreasing growth in others. Together, our results reveal new groups of microalgal exometabolites, some of which could alter algal growth when provided exogenously, suggesting potential roles in allelopathy and algal interactions.

A New, Quick, and Simple Protocol to Evaluate Microalgae Polysaccharide Composition

In this work, a new methodological approach, relying on the high specificity of enzymes in a complex mixture, was developed to estimate the composition of bioactive polysaccharides produced by microalgae, directly in algal cultures. The objective was to set up a protocol to target oligomers commonly known to be associated with exopolysaccharides’ (EPS) nutraceutical and pharmaceutical activities (i.e., rhamnose, fucose, acidic sugars, etc.) without the constraints classically associated with chromatographic methods, while maintaining a resolution sufficiently high to enable their monitoring in the culture system. Determination of the monosaccharide content required the application of acid hydrolysis (2 M trifluoroacetic acid) followed by NaOH (2 M) neutralization. Quantification was then carried out directly on the fresh hydrolysate using enzyme kits corresponding to the main monosaccharides in a pre-determined composition of the polysaccharides under analysis. Initial results showed that the enzymes were not sensitive to the presence of TFA and NaOH, so the methodology could be carried out on fresh hydrolysate. The limits of quantification of the method were estimated as being in the order of the log of nanograms of monosaccharides per well, thus positioning it among the chromatographic methods in terms of analytical performance. A comparative analysis of the results obtained by the enzymatic method with a reference method (high-performance anion-exchange chromatography) confirmed good recovery rates, thus validating the closeness of the protocol. Finally, analyses of raw culture media were carried out and compared to the results obtained in miliQ water; no differences were observed. The new approach is a quick, functional analysis method allowing routine monitoring of the quality of bioactive polysaccharides in algal cultures grown in photobioreactors.

The Use of Algae in the Process of Cadmium and Lead Ions Removal from Wastewater

The study presents the possibility of using chlorophyta in the removal of cadmium and lead ions from industrial wastewater produced after the washing of equipment used in the manufacture of battery and batteries. The process was conducted with the use of two algal cultures: Raphidocelis subcapitata produced in laboratory conditions, and a mixed chlorophyta population collected from a natural, eutrophicated water reservoir with heavy metal ions present in the water and sludge. The study showed that the effectiveness of a pure algal culture is comparable to that of a mixed chlorophyta population, characterized by greater diversity of functional groups at binding sites and higher resistance to stress that may occur in the wastewater environment. The maximum effectiveness of ions sorption was 64% for cadmium (mixed algal population) and 60% for lead (Raphidocelis subcapitata).

Effects of Co-culture on Improved Productivity and Bioresource for Microalgal Biomass Using the Floc-Forming Bacteria Melaminivora Jejuensis

Bacterial and algal floc formation was induced by inoculating three species of wastewater-derived bacteria (Melaminivora jejuensis, Comamonas flocculans, and Escherichia coli) into algal cultures (Chlorella sorokiniana). Bacterial and algal flocs formed in algal cultures inoculated with M. jejuensis and C. flocculans, and these flocs showed higher sedimentation rates than pure algal culture. The floc formed by M. jejuensis (4988.46 ± 2589.81 μm) was 10-fold larger than the floc formed by C. flocculans (488.60 ± 226.22 μm), with a three-fold higher sedimentation rate (M. jejuensis, 91.08 ± 2.32% and C. flocculans, 32.55 ± 6.33%). Biomass and lipid productivity were improved with M. jejuensis inoculation [biomass, 102.25 ± 0.35 mg/(L·day) and 57.80 ± 0.20 mg/(L·day)] compared with the productivity obtained under pure algal culture conditions [biomass, 78.00 ± 3.89 mg/(L·day) and lipids, 42.26 ± 2.11 mg/(L·day)]. Furthermore, the fatty acid composition of the biomass produced under pure algal culture conditions was mainly composed of C16:0 (43.67%) and C18:2 (45.99%), whereas the fatty acid composition of the biomass produced by M. jejuensis was mainly C16:0 (31.80%), C16:1 (24.45%), C18:1 (20.23%), and C18:2 (16.11%). These results suggest the possibility of developing an efficient method for harvesting microalgae using M. jejuensis and provide information on how to improve biomass productivity using floc-forming bacteria.

The ice-nucleating activity of Arctic sea surface microlayer samples and marine algal cultures

In recent years, sea spray as well as the biological material it contains has received increased attention as a source of ice-nucleating particles (INPs). Such INPs may play a role in remote marine regions, where other sources of INPs are scarce or absent. In the Arctic, these INPs can influence water–ice partitioning in low-level clouds and thereby the cloud lifetime, with consequences for the surface energy budget, sea ice formation and melt, and climate. Marine aerosol is of a diverse nature, so identifying sources of INPs is challenging. One fraction of marine bioaerosol (phytoplankton and their exudates) has been a particular focus of marine INP research. In our study we attempt to address three main questions. Firstly, we compare the ice-nucleating ability of two common phytoplankton species with Arctic seawater microlayer samples using the same instrumentation to see if these phytoplankton species produce ice-nucleating material with sufficient activity to account for the ice nucleation observed in Arctic microlayer samples. We present the first measurements of the ice-nucleating ability of two predominant phytoplankton species: Melosira arctica, a common Arctic diatom species, and Skeletonema marinoi, a ubiquitous diatom species across oceans worldwide. To determine the potential effect of nutrient conditions and characteristics of the algal culture, such as the amount of organic carbon associated with algal cells, on the ice nucleation activity, Skeletonema marinoi was grown under different nutrient regimes. From comparison of the ice nucleation data of the algal cultures to those obtained from a range of sea surface microlayer (SML) samples obtained during three different field expeditions to the Arctic (ACCACIA, NETCARE, and ASCOS), we found that they were not as ice active as the investigated microlayer samples, although these diatoms do produce ice-nucleating material. Secondly, to improve our understanding of local Arctic marine sources as atmospheric INPs we applied two aerosolization techniques to analyse the ice-nucleating ability of aerosolized microlayer and algal samples. The aerosols were generated either by direct nebulization of the undiluted bulk solutions or by the addition of the samples to a sea spray simulation chamber filled with artificial seawater. The latter method generates aerosol particles using a plunging jet to mimic the process of oceanic wave breaking. We observed that the aerosols produced using this approach can be ice active, indicating that the ice-nucleating material in seawater can indeed transfer to the aerosol phase. Thirdly, we attempted to measure ice nucleation activity across the entire temperature range relevant for mixed-phase clouds using a suite of ice nucleation measurement techniques – an expansion cloud chamber, a continuous-flow diffusion chamber, and a cold stage. In order to compare the measurements made using the different instruments, we have normalized the data in relation to the mass of salt present in the nascent sea spray aerosol. At temperatures above 248 K some of the SML samples were very effective at nucleating ice, but there was substantial variability between the different samples. In contrast, there was much less variability between samples below 248 K. We discuss our results in the context of aerosol–cloud interactions in the Arctic with a focus on furthering our understanding of which INP types may be important in the Arctic atmosphere.