Physiological Limitations and Solutions to Various Applications of Microalgae

Despite over a century of research, the various applications of microalgae have only been realized only since the 1940s. With a repertoire of valuable products like biodiesel, astaxanthin, canthaxanthin, lutein, β-carotene, phycocyanin, chlorophyll a, polyunsaturated fatty acids, exopolymeric substance (EPS), and biohydrogen, the commercial importance of microalgae and demand for its product is gaining increasing attention. However, successful transition of the synthesis of microalgal products from laboratory to industries has yet to be realized, even after over 70 years of extensive research. This failure of commercial success of microalgal products can be attributed to the lack of understanding of the physiological role of the products and biological constraint placed by the bioenergetics and physiology, which has been largely ignored. This chapter focuses on the physiological limitations behind synthesis of microalgal products, highlights the crucial unknowns behind the role and synthesis of these products, and hints strategies to overcome the limitations to realize the commercial dream of microalgal products

Core metabolism plasticity in phytoplankton: Response of Dunaliella tertiolecta to oil exposure

Human alterations to the marine environment such as an oil spill can induce oxidative stress in phytoplankton. Exposure to oil has been shown to be lethal to most phytoplankton species, but some are able to survive and grow at unaffected or reduced growth rates, which appears to be independent of the class and phylum of the phytoplankton and their ability to consume components of oil heterotrophically. The goal of this article is to test the role of core metabolism plasticity in the oil-resisting ability of phytoplankton. Experiments were performed on the oil- resistant chlorophyte, Dunaliella tertiolecta, in control and water accommodated fractions of oil, with and without metabolic inhibitors targeting the core metabolic pathways. We observed that inhibiting pathways such as photosynthetic electron transport (PET) and pentose-phosphate pathway were lethal; however, inhibition of pathways such as mitochondrial electron transport and cyclic electron transport caused growth to be arrested. Pathways such as photorespiration and Kreb\u27s cycle appear to play a critical role in the oil-tolerating ability of D. tertiolecta. Analysis of photo-physiology revealed reduced PET under inhibition of photorespiration but not Kreb\u27s cycle. Further studies showed enhanced flux through Kreb\u27s cycle suggesting increased energy production and photorespiration counteract oxidative stress. Lastly, reduced extracellular carbohydrate secretion under oil exposure indicated carbon and energy conservation, which together with enhanced flux through Kreb\u27s cycle played a major role in the survival of D. tertiolecta under oil exposure by meeting the additional energy demands. Overall, we present data that suggest the role of phenotypic plasticity of multiple core metabolic pathways in accounting for the oxidative stress tolerating ability of certain phytoplankton species

Molecular mechanism of oil induced growth inhibition in diatoms using Thalassiosira pseudonana as the model species

The 2010 Deepwater Horizon oil-spill exposed the microbes of Gulf of Mexico to unprecedented amount of oil. Conclusive evidence of the underlying molecular mechanism(s) on the negative effects of oil exposure on certain phytoplankton species such as Thalassiosira pseudonana is still lacking, curtailing our understanding of how oil spills alter community composition. We performed experiments on model diatom T. pseudonana to understand the mechanisms underpinning observed reduced growth and photosynthesis rates during oil exposure. Results show severe impairment to processes upstream of photosynthesis, such as light absorption, with proteins associated with the light harvesting complex damaged while the pigments were unaffected. Proteins associated with photosynthetic electron transport were also damaged, severely affecting photosynthetic apparatus and depriving cells of energy and carbon for growth. Negative growth effects were alleviated when an organic carbon source was provided. Further investigation through proteomics combined with pathway enrichment analysis confirmed the above findings, while highlighting other negatively affected processes such as those associated with ferroxidase complex, high-affinity iron-permease complex, and multiple transmembrane transport. We also show that oxidative stress is not the primary route of negative effects, rather secondary. Overall, this study provides a mechanistic understanding of the cellular damage that occurs during oil exposure to T. pseudonana

Aggregation and Degradation of Dispersants and Oil by Microbial Exopolymers (ADDOMEx): Toward a Synthesis of Processes and Pathways of Marine Oil Snow Formation in Determining the Fate of Hydrocarbons

Microbes (bacteria, phytoplankton) in the ocean are responsible for the copious production of exopolymeric substances (EPS) that include transparent exopolymeric particles. These materials act as a matrix to form marine snow. After the Deepwater Horizon oil spill, marine oil snow (MOS) formed in massive quantities and influenced the fate and transport of oil in the ocean. The processes and pathways of MOS formation require further elucidation to be better understood, in particular we need to better understand how dispersants affect aggregation and degradation of oil. Toward that end, recent work has characterized EPS as a function of microbial community and environmental conditions. We present a conceptual model that incorporates recent findings in our understanding of the driving forces of MOS sedimentation and flocculent accumulation (MOSSFA) including factors that influence the scavenging of oil into MOS and the routes that promote decomposition of the oil post MOS formation. In particular, the model incorporates advances in our understanding of processes that control interactions between oil, dispersant, and EPS in producing either MOS that can sink or dispersed gels promoting microbial degradation of oil compounds. A critical element is the role of protein to carbohydrate ratios (P/C ratios) of EPS in the aggregation process of colloid and particle formation. The P/C ratio of EPS provides a chemical basis for the stickiness ; factor that is used in analytical or numerical simulations of the aggregation process. This factor also provides a relative measure for the strength of attachment of EPS to particle surfaces. Results from recent laboratory experiments demonstrate (i) the rapid formation of microbial assemblages, including their EPS, on oil droplets that is enhanced in the presence of Corexit-dispersed oil, and (ii) the subsequent rapid oil oxidation and microbial degradation in water. These findings, combined with the conceptual model, further improve our understanding of the fate of the sinking MOS (e.g., subsequent sedimentation and preservation/degradation) and expand our ability to predict the behavior and transport of spilled oil in the ocean, and the potential effects of Corexit application, specifically with respect to MOS processes (i.e., formation, fate, and half-lives) and Marine Oil Snow Sedimentation and Flocculent Accumulation

Molecular Nature of Marine Particulate Organic Iron-Carrying Moieties Revealed by Electrospray Ionization Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry (ESI-FTICRMS)

Marine sinking particulate organic matter (POM), acting as a link between surface primary production and burial of organic matter in marine sediments, undergoes a variety of physical and biochemical alterations on its way to the deep ocean, resulting in an increase in its un-characterizable proportion with diagenesis. Further, the binding ligands in POM for iron, an essential nutrient to marine life and tightly coupled with organic matter, has rarely been studied. In the current study, we employed an approach combining sequential extraction with ultrahigh resolution mass spectrometry (ESI-FTICRMS), in order to explore and unravel the chemical characteristics of organic matter compounds relevant to marine particle flux within the mesopelagic and deep ocean, with a focus on the potential iron-carrying molecules. With increasing depth, POM increases in aliphaticity, and decreases in intensity-normalized O/C ratios, aromatics, and carboxylic-rich alicyclic molecules (CRAM)-like compounds. The potential iron-carrying molecules account for ∼14% of total identified molecules, and appear to have been incorporated into the marine particles via ion complexation, hydrophobic interaction, and/or interlayered “occlusion.” The relative abundance of iron-binding organic molecules in these three operationally-defined categories changes with depth: “surficially-complexed” fraction decreases with depth, the “interlayered-occluded” fraction increases to a comparable extent and “hydrophobic interaction” fraction occurs at all depths. Collectively, the potential iron-carrying organic molecules exhibit a set of unique molecular characteristics: a relatively lower average H/C ratio and a higher O/C ratio compared to bulk POM, a dominance of aromatics, black carbon-like compounds and CRAM-like compounds, and minor amounts of aliphatics. These molecules exhibit partial similar molecular features as precursors formed from photochemical reactions in the surface ocean, but they have been greatly modified by flux processes. Noticeably, a minor fraction of these iron-carrying molecules

The Role of Microbial Exopolymers in Determining the Fate of Oil and Chemical Dispersants in the Ocean

The production of extracellular polymeric substances (EPS) by planktonic microbes can influence the fate of oil and chemical dispersants in the ocean through emulsification, degradation, dispersion, aggregation, and/or sedimentation. In turn, microbial community structure and function, including the production and character of EPS, is influenced by the concentration and chemical composition of oil and chemical dispersants. For example, the production of marine oil snow and its sedimentation and flocculent accumulation to the seafloor were observed on an expansive scale after the Deepwater Horizon oil spill in the Northern Gulf of Mexico in 2010, but little is known about the underlying control of these processes. Here, we review what we do know about microbially produced EPS, how oil and chemical dispersant can influence the production rate and chemical and physical properties of EPS, and ultimately the fate of oil in the water column. To improve our response to future oil spills, we need a better understanding of the biological and physiochemical controls of EPS production by microbes under a range of environmental conditions, and in this paper, we provide the key knowledge gaps that need to be filled to do so

Tumor-induced osteomalacia: experience from three tertiary care centers in India

Tumor-induced osteomalacia (TIO) is a rare paraneoplastic syndrome characterized by recalcitrant hypophosphatemia. Reports from the Indian subcontinent are scarce, with most being single center experiences involving few patients. Herein, we conducted a retrospective analysis of 30 patients of TIO diagnosed at three tertiary care hospitals in India. Patients with persistent hypophosphatemia (despite correction of hypovitaminosis D), normocalcemia, elevated alkaline phosphatase, low TmP/GFR and elevated or ‘inappropriately normal’ FGF23 levels were labeled as having TIO. They were sequentially subjected to functional followed by anatomical imaging. Patients with a well-localized tumor underwent excision; others were put on phosphorous and calcitriol supplementation. The mean age at presentation was 39.6 years with female:male ratio of 3:2. Bone pain (83.3%) and proximal myopathy (70%) were the chief complaints; 40% of cases had fractures. The mean delay in diagnosis was 3.8 years. Tumors were clinically detectable in four patients (13.3%). The mean serum phosphate was 0.50 mmol/L with a median serum FGF23 level of 518 RU/mL. Somatostatin receptor-based scintigraphy was found to be superior to FDG-PET in tumor localization. Lower extremities were the most common site of the tumor (72%). Tumor size was positively correlated with serum FGF23 levels. Twenty-two patients underwent tumor resection and 16 of them had phosphaturic mesenchymal tumors. Surgical excision led to cure in 72.7% of patients whereas disease persistence and disease recurrence were seen in 18.2% and 9.1% of cases, respectively. At the last follow-up, serum phosphate in the surgically treated group was significantly higher than in the medically managed group

Optimization of microalgae for enhanced lipid production

The ever-increasing human demand for energy is leading to depletion of the planet’s fossil fuel reserves. Past records have shown that the price of fossil fuel has always been increasing and it is expected to rise continuously with demand in future. Use of fossil fuel generates significant amounts of greenhouse gases, which are contributing to climate change. These factors clearly highlight the need of a cheaper, renewable, and an eco-friendly fuel source. Past research has suggested that biodiesel from algae could be a possible alternative, as microalgae have been shown to produce the highest lipid yield in the lowest area when compared to other biological sources. However, despite having a high lipid yield per area, biodiesel produced by using the lipids from growing algae using current cultivation methods will not be able to meet the increasing energy demands. Furthermore, the cost of algal biomass production is very high for economic implementation on an industrial scale. Therefore the research reported here focuses on optimizing microalgal performance for biodiesel production, both phototrophically and heterotrophically. Under phototrophic conditions we analysed three different approaches to optimise lipid production; use of chemical inhibitors, phosphorus starvation and combined nitrogen and phosphorus starvation. Methionine sulfoximine (MSX) was used as a chemical inhibitor for nitrogen assimilation for instant nitrogen starvation in cultures, as nitrogen starvation or limitation has been shown to increase the lipid content of microalgae. Use of MSX induced rapid lipid accumulation in cells and prolonged higher lipid content (2-fold higher) in cells as compared to the controls. Phosphorus starvation of cells led to lower biomass but showed up to three-fold higher cellular lipid content without affecting the photosynthetic efficiency. Combined nitrogen and phosphorus starvation also resulted in lower biomass but higher cellular lipid content; however combined nitrogen and phosphorus starvation had no synergistic effect on the lipid synthesis over individually starved nitrogen or phosphorus conditions. Also, comparison of the parameters measuring the photosynthetic physiology implied that nitrogen starvation had a more dominant effect on the photosynthetic performance of the cells than phosphorus starvation in the combined nutrient starved group. As a result, the combined nitrogen and phosphorus-starved cells behaved similarly to the nitrogen starved rather than the phosphorusstarved cells in terms of photosynthetic performance. Overall all the tested condition resulted in higher cellular neutral lipid as compared to their own control group, however the neutral lipid measured per ml was still similar to the control groups. This was mainly due to the lower biomass generated under nutrient limited/starved conditions. Therefore, investigation of other modes of cultivation such as heterotrophy and mixotrophy was done to overcome this problem. Scenedesmus sp. was used for heterotrophic studies using molasses as the carbon source in growth media. Under heterotrophic conditions, Scenedesemus sp. performed much better than in phototrophic conditions in terms of both biomass (2.5-fold higher) and much higher lipid content (neutral lipid per ml). Surprisingly, the heterotrophically grown Scenedesmus sp. still retained their chlorophyll in the dark, indicating the possibility of retention of active photosynthetic machinery (5 ng chlorophyll per cell was measured in heterotrophic cells). Exposure of stationary phase heterotrophically grown cells to light caused stimulation of growth that in turn increased the biomass and total neutral lipid content even further. Low rates of oxygen evolution (0.2 femtomol O2.min-1.μm-3) suggested the presence of an active photosynthetic apparatus in heterotrophically grown cells, which might have played a significant role in the observed growth stimulation in stationary phase cells. Further investigations revealed an interesting set of changes to the photosynthetic apparatus in heterotrophically grown cells, including more than two-fold lower absorption cross-sectional area and NPQ values, higher electron transport flux, and two-fold higher connectivity between reaction centers as compared to the photosynthetically grown cultures. Eventually, when all the tested methods were compared with respect to the total neutral lipid per ml, neutral lipid productivity, and final cell number normalised to the values obtained under the control conditions of the respective experiments, we found a combination of heterotrophy followed by mixotrophy is likely to be a better way of producing algal biomass for the production of biodiesel and other co-products

Optimization of microalgae for enhanced lipid production

The ever-increasing human demand for energy is leading to depletion of the planet’s fossil fuel reserves. Past records have shown that the price of fossil fuel has always been increasing and it is expected to rise continuously with demand in future. Use of fossil fuel generates significant amounts of greenhouse gases, which are contributing to climate change. These factors clearly highlight the need of a cheaper, renewable, and an eco-friendly fuel source. Past research has suggested that biodiesel from algae could be a possible alternative, as microalgae have been shown to produce the highest lipid yield in the lowest area when compared to other biological sources. However, despite having a high lipid yield per area, biodiesel produced by using the lipids from growing algae using current cultivation methods will not be able to meet the increasing energy demands. Furthermore, the cost of algal biomass production is very high for economic implementation on an industrial scale. Therefore the research reported here focuses on optimizing microalgal performance for biodiesel production, both phototrophically and heterotrophically. Under phototrophic conditions we analysed three different approaches to optimise lipid production; use of chemical inhibitors, phosphorus starvation and combined nitrogen and phosphorus starvation. Methionine sulfoximine (MSX) was used as a chemical inhibitor for nitrogen assimilation for instant nitrogen starvation in cultures, as nitrogen starvation or limitation has been shown to increase the lipid content of microalgae. Use of MSX induced rapid lipid accumulation in cells and prolonged higher lipid content (2-fold higher) in cells as compared to the controls. Phosphorus starvation of cells led to lower biomass but showed up to three-fold higher cellular lipid content without affecting the photosynthetic efficiency. Combined nitrogen and phosphorus starvation also resulted in lower biomass but higher cellular lipid content; however combined nitrogen and phosphorus starvation had no synergistic effect on the lipid synthesis over individually starved nitrogen or phosphorus conditions. Also, comparison of the parameters measuring the photosynthetic physiology implied that nitrogen starvation had a more dominant effect on the photosynthetic performance of the cells than phosphorus starvation in the combined nutrient starved group. As a result, the combined nitrogen and phosphorus-starved cells behaved similarly to the nitrogen starved rather than the phosphorusstarved cells in terms of photosynthetic performance. Overall all the tested condition resulted in higher cellular neutral lipid as compared to their own control group, however the neutral lipid measured per ml was still similar to the control groups. This was mainly due to the lower biomass generated under nutrient limited/starved conditions. Therefore, investigation of other modes of cultivation such as heterotrophy and mixotrophy was done to overcome this problem. Scenedesmus sp. was used for heterotrophic studies using molasses as the carbon source in growth media. Under heterotrophic conditions, Scenedesemus sp. performed much better than in phototrophic conditions in terms of both biomass (2.5-fold higher) and much higher lipid content (neutral lipid per ml). Surprisingly, the heterotrophically grown Scenedesmus sp. still retained their chlorophyll in the dark, indicating the possibility of retention of active photosynthetic machinery (5 ng chlorophyll per cell was measured in heterotrophic cells). Exposure of stationary phase heterotrophically grown cells to light caused stimulation of growth that in turn increased the biomass and total neutral lipid content even further. Low rates of oxygen evolution (0.2 femtomol O2.min-1.μm-3) suggested the presence of an active photosynthetic apparatus in heterotrophically grown cells, which might have played a significant role in the observed growth stimulation in stationary phase cells. Further investigations revealed an interesting set of changes to the photosynthetic apparatus in heterotrophically grown cells, including more than two-fold lower absorption cross-sectional area and NPQ values, higher electron transport flux, and two-fold higher connectivity between reaction centers as compared to the photosynthetically grown cultures. Eventually, when all the tested methods were compared with respect to the total neutral lipid per ml, neutral lipid productivity, and final cell number normalised to the values obtained under the control conditions of the respective experiments, we found a combination of heterotrophy followed by mixotrophy is likely to be a better way of producing algal biomass for the production of biodiesel and other co-products