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Survival of the fattest
Abstract
Large-scale production of energetic storage compounds by microalgae is hampered by competition and evolution. Both phenomena result in contamination and arise due to a mismatch between the desired productive microalgal strain and the constructed environment. The prevailing approach to solve this issue involves increasing the survival potential of the desired strain, for example by working in closed systems or at extreme conditions. We advocate adjusting the environment in such a way that lipid production, or any other desired characteristic, gives a competitive advantage. Competition and evolution become a value rather than a threat to processes in which the desired characteristic is ensured by a selective environment. Research and cultivation efforts will benefit from this approach as it harnesses the microalgal diversity in nature.
... This is a particularly interesting characteristic for algal biodiesel production. Therefore, some researchers suggest that selection for low-density cells would lead to the dominance of lipid-producing microalgal strains under mixed culture conditions [11,12]. Conceivably, selective enrichment of auto-floating microalgae would be a promising strategy to simultaneously overcome the problems associated with harvesting and culture control. ...
... The above results indicate that a short separation time (5 min) was not appropriate to induce autoflotation, although it was expected to create a strong selective pressure towards the selection of low-density microalgal cells [11,12]. Correspondingly, the separation time was increased step-wise to 60 min within one week in Period III, and instant enhancement of autoflotation was observed. ...
... In the present system, most nitrate was assimilated in the dark phase (Dark end, Fig. 6a). Previous studies reported that assimilation in the dark is driven by the presence of storage compounds (e.g., carbohydrates, lipids) [12,36]. To confirm this, a cyclic study was conducted on day 244 in Period IV (Fig. 7). ...
Integrating microalgal cultivation with wastewater treatment is a promising option for sustainable resource recovery and eco-friendly wastewater bioremediation. However, harvesting and culture control are major hurdles hindering its application. This study provided an effective solution to these challenges by selective enrichment of auto-floating microalgae in mixed cultures. This was achieved by repeatedly retaining the floe layer and discharging the subnatant in a semi-batch photo-bioreactor. The enriched auto-floating microalgae were dominated by Tribonema sp., which maintained its dominance (84.8 ± 9.2 %) over 180 days under non-sterile conditions. Effective autoflotation (>90 %) was achieved at a separation time of 30 min without coagulation, which enabled cost-effective microalgae harvesting. The autoflotation mechanism was in situ gas flotation, induced by photosynthetic micro‑oxygen bubbles. Cell surface hydrophobicity played a pivotal role in effective bubble-cell adhesion. This autoflotation mechanism inherently led to excellent dewaterability (SRF = 2.7 × 10¹¹ m/kg). The enriched auto-floating microalgae demonstrated excellent nutrient removal efficiency (N and P > 98 %), and high biodiesel production potential (TFA content: 27–29 %; biodiesel productivity: 48–76 mg/L/d). It also had a high content (29–31 %) of β-(1–3/1–6)-glucans, which are well-known biological response modifiers and have wide applications in pharmaceuticals, nutraceuticals, food, feed, and cosmetics sectors. Overall, the present work demonstrates that selective enrichment of auto-floating microalgae offers great opportunities for a microalgae-based biorefinery in sustainable wastewater bioremediation.
... In these conditions the accumulation ability for lipids or polysaccharides is not a competitive advantage. Therefore, when microorganisms with high accumulation ability are cultivated in culture medium with high nutrient concentration, culture contaminations by unproductive cells (low accumulation ability, but higher grow rate) can rapidly replace the productive strains (Chubukov et al., 2017;Di Caprio et al., 2019;Mooij et al., 2015bMooij et al., , 2013. In this work the word "contaminant" is used to indicate every microbial cell, in the culture medium, with an accumulation ability lower than the target strain cultivated. ...
... These processes are usually based on the alternance of periods in which substrate (energy/C source) is available, named feast phase, and periods in which substrate is no longer available, named famine phase. The aim of these processes is pushing towards the survival of the "fattest" strains in place of the survival of the sole fastest strains (> μ max ) (Mooij et al., 2015b(Mooij et al., , 2013, otherwise obtained with culture medium fully replete in every nutrient. ...
... The general approach used is to alternate an energy replete condition with an energy starvation condition, to select those strains with more accumulated storage compounds (Di Caprio et al., 2019;Mooij et al., 2015b;Oliveira et al., 2017). This concept can be synthetized as the survival of the "fattest" (Mooij et al., 2015b(Mooij et al., , 2013 in place of the common survival of the "fastest" (Fig. 2). ...
The microbial ability to accumulate biomolecules is fundamental for different biotechnological applications aiming at the production of biofuels, food and bioplastics. However, high accumulation is a selective advantage only under certain stressful conditions, such as nutrient depletion, characterized by lower growth rate. Conventional bioprocesses maintain an optimal and stable environment for large part of the cultivation, that doesn't reward cells for their accumulation ability, raising the risk of selection of contaminant strains with higher growth rate, but lower accumulation of products.
Here in this work the physiological responses of different microorganisms (microalgae, bacteria, yeasts) under N-starvation and energy starvation are reviewed, with the aim to furnish relevant insights exploitable to develop tailored bioprocesses to select specific strains for their higher accumulation ability.
Microorganism responses to starvation are reviewed focusing on cell cycle, biomass production and variations in biochemical composition. Then, the work describes different innovative bioprocess configurations exploiting uncoupled nutrient feeding strategies (feast-famine), tailored to maintain a selective pressure to reward the strains with higher accumulation ability in mixed microbial populations. Finally, the main models developed in recent studies to describe and predict microbial growth and intracellular accumulation upon N-starvation and feast-famine conditions have been reviewed.
... However, maintaining a large-scale axenic phototrophic system is expensive and cumbersome [48]. Instead, we propose to grow microalgae in an open system and to use selective pressure to enrich algae with a high storage compound production capacity, as described by Mooij et al. [49]. In their approach, Mooij et al. [49] subjected an open algal culture in a photobioreactor to a cyclical light/dark regimen. ...
... Instead, we propose to grow microalgae in an open system and to use selective pressure to enrich algae with a high storage compound production capacity, as described by Mooij et al. [49]. In their approach, Mooij et al. [49] subjected an open algal culture in a photobioreactor to a cyclical light/dark regimen. During the light period carbon dioxide was supplied, while nitrogen was supplied only during the dark period. ...
... A separation step after the anaerobic digester should prevent solids from entering the phototrophic system; otherwise, the turbidity in the tank would become too high and prevent light from reaching the algal biofilm. the polyglucose level in the biomass produced by Mooij et al. [49] was comparable with the highest reported values for pure cultures (57 AE 2% of volatile suspended solids). ...
Microalgal biomass can be converted to biofuels to replace nonsustainable fossil fuels, but the widespread use of microalgal biofuels remains hampered by the high energetic and monetary costs related to carbon dioxide supply and downstream processing. Growing microalgae in mixed culture biofilms reduces energy demands for mixing, maintaining axenic conditions, and biomass concentration. Furthermore, maintaining a high pH improves carbon dioxide absorption rates and inorganic carbon solubility, thus overcoming the carbon limitation and increasing the volumetric productivity of the microalgal biomass. Digesting the microalgal biomass anaerobically at high pH results in biogas that is enriched in methane, while the dissolved carbon dioxide is recycled to the phototrophic reactor. All of the required haloalkaline conversions are known in nature.
... To increase competitiveness, the use of waste substrates must be combined with mixed culture biotechnology and engineering designs with reduced costs and easily maintained operational conditions (Gujjala et al., 2019;Kourmentza et al., 2017;Kumar et al., 2020;Yadav et al., 2020). This solution considers engineering the ecosystem rather than specific strains to maintain a characteristic or functionality in the culture (Kourmentza et al., 2017;Mooij et al., 2013). Mooij et al. (2013) showed how to obtain a stable, open system enriched in storing populations inspired by Darwin's Theory of evolution by natural selection. ...
... This solution considers engineering the ecosystem rather than specific strains to maintain a characteristic or functionality in the culture (Kourmentza et al., 2017;Mooij et al., 2013). Mooij et al. (2013) showed how to obtain a stable, open system enriched in storing populations inspired by Darwin's Theory of evolution by natural selection. To design better biotechnologies, they proposed that imposing the right selective environment can emphasize the ecological role of storage compounds, allowing for the robust enrichment of a complex culture with optimal storage capacity. ...
Operational conditions select for preferent TAG or PHA storage from waste lipids. • Coupled C and N supply promotes TAG production. • Uncoupled C and N feedings along with limited C excess favours PHA over TAG storage. • Pathways analysis unravelled the experimentally slower synthesis of PHA than TAG. Editor: Daniel CW Tsang The lipid fraction of the effluents generated in several food-processing activities can be transformed into polyhydroxyalkanoates (PHAs) and triacylglycerides (TAGs), through open culture biotechnologies. Although competition between storing and non-storing populations in mixed microbial cultures (MMCs) has been widely studied, the right selective environment allowing for the robust enrichment of a community when different types of accumulators coexist is still not clear. In this research, comprehensive metabolic analyses of PHA and TAG synthesis and degradation, and concomitant respiration of external carbon, were used to understand and explain the changes observed in a laboratory-scale bioreactor fed with the lipid-rich fraction (mainly oleic acid) of a waste-water stream produced in the fish-canning industry. It was concluded that the mode of oxygen, carbon, and nitrogen supply determines the enrichment of the culture in specific populations, and hence the type of intracellular compounds preferentially accumulated. Coupled carbon and nitrogen feeding regime mainly selects for TAG producers whereas uncoupled feeding leads to PHA or TAG production function of the rate of carbon supply under specific aeration rates and feast and famine phases lengths.
... Furthermore, the potential for outdoors wastewater treatment (Ledda et al., 2015) and abatement performance of CO 2 and toluene contained in exhaust gas by marine microalgae using diluted centrate in seawater as nutrient source (Anbalagan et al., 2017) have been recently addressed in closed photobioreactors. On the other hand, this photosynthetic CO 2abatement process can be further optimized by implementing the novel nutrient supplementation strategy developed by Mooij et al. (2013) to promote the cyclic production of storage compounds in the algal biomass. In this context, the production of a biomass with a high content in the metabolites of interest will increase the economic sustainability of the process (Toledo-Cervantes et al., 2017a). ...
... However, it is important to highlight that this operation is typically performed batch-wise in two operation stages with different nutrient requirements: a preliminary biomass production stage is necessary before inducing carbohydrates accumulation in the second stage. In this sense, a novel strategy of feeding nitrogen only during the dark period was here applied as a strategy to induce the continuous accumulation of high-energy storage compounds (Mooij et al., 2013). It is worthy to highlight that this strategy has been implemented exclusively in stirred reactors operated in sequencing batch mode (Mooij et al., 2015). ...
The long-term performance of a tubular photobioreactor interconnected to a gas absorption column for the abatement of CO2 from biogas and flue-gas was investigated. Additionally, a novel nitrogen feast-famine regime was implemented during the flue-gas feeding stage in order to promote the continuous storage of highly-energetic compounds. Results showed effective CO2 (~98%) and H2S (~99%) removals from synthetic biogas, supported by the high photosynthetic activity of microalgae which resulted in an alkaline pH (~10). In addition, CO2 removals of 99 and 91% were observed during the flue-gas operation depending on the nutrients source: mineral salt medium and digestate, respectively. A biomass productivity of ~8 g m⁻² d⁻¹ was obtained during both stages, with a complete nitrogen and carbon recovery from the cultivation broth. Moreover, the strategy of feeding nutrients during the dark period promoted the continuous accumulation of carbohydrates, their concentration increasing from 22% under normal nutrition up to 37% during the feast-famine cycle. This represents a productivity of ~3 g-carbohydrates m⁻² d⁻¹, which can be further valorized to contribute to the economic sustainability of the photosynthetic CO2 removal process.
... Microalgae are unicellular and photosynthetic microorganisms, ranging from 0.2 to 2 μm (picoplankton) up to filamentous forms with sizes of 100 μm or higher ( Figure 2) [5][6][7]. These are unicellular organisms that consist of both prokaryotic (Cyanophyceae) or eukaryotic (Chlorophyta) organisms and they can grow rapidly in aquatic environments such as fresh water, waste water, and the marine environment. ...
... Microalgae are unicellular and photosynthetic microorganisms, ranging from 0.2 to 2 µm (picoplankton) up to filamentous forms with sizes of 100 µm or higher ( Figure 2) [5][6][7]. These are unicellular organisms that consist of both prokaryotic (Cyanophyceae) or eukaryotic (Chlorophyta) organisms and they can grow rapidly in aquatic environments such as fresh water, waste water, and the marine environment. ...
Substantial progress has been made in algal technologies in past few decades. Initially, microalgae drew the attention of the scientific community as a renewable source of biofuels due to its high productivity over a short period of time and potential of significant lipid accumulation. As of now, a technological upsurge has elaborated its scope in phycoremediation of both organic and inorganic pollutants. The dual role of microalgae—i.e., phycoremediation coupled with energy production—is well established, however, commercially, algal biofuel production is not yet sustainable due to high energy inputs. Efforts are being made to make the algal biofuel economy through modification in the cultivation conditions, harvesting, and extraction of value added products. Recent studies have demonstrated algal biomass production with various types of wastewater and industrial effluents. Similarly, the recent advent of eco-friendly harvesting technologies—such as low-cost green coagulants, electrochemical harvesting, etc.—are energy efficient and economical. Contemporary improvement in efficient lipid extraction from biomass will make algal biodiesel economical. The absolute extraction of all the value added products from algal biomass, either whole cell or lipid extracted biomass, in a complete biorefinery approach will be more economical and eco-friendly.
... Using plants for the production of chemicals will require massive amounts of land, ultimately resulting in competition between biomass production, agriculture and nature. Therefore, microorganisms, and more specifically photosynthetic microalgae, can be used for the production of starch, carotenoids, fatty acids, proteins, antioxidants and pigments (Mooij et al., 2013;Gangl et al., 2015). Fixing CO 2 to produce larger biomolecules requires energy. ...
Production of organic molecules is largely depending on fossil fuels. A sustainable alternative would be the synthesis of these compounds from CO2 and a cheap energy source, such as H2, CH4, NH3, CO, sulfur compounds or iron(II). Volcanic and geothermal areas are rich in CO2 and reduced inorganic gasses and therefore habitats where novel chemolithoautotrophic microorganisms for the synthesis of organic compounds could be discovered. Here we describe “Candidatus Hydrogenisulfobacillus filiaventi” R50 gen. nov., sp. nov., a thermoacidophilic, autotrophic H2-oxidizing microorganism, that fixed CO2 and excreted no less than 0.54 mol organic carbon per mole fixed CO2. Extensive metabolomics and NMR analyses revealed that Val, Ala and Ile are the most dominant form of excreted organic carbon while the aromatic amino acids Tyr and Phe, and Glu and Lys were present at much lower concentrations. In addition to these proteinogenic amino acids, the excreted carbon consisted of homoserine lactone, homoserine and an unidentified amino acid. The biological role of the excretion remains uncertain. In the laboratory, we noticed the production under high growth rates (0.034 h⁻¹, doubling time of 20 h) in combination with O2-limitation, which will most likely not occur in the natural habitat of this strain. Nevertheless, this large production of extracellular organic molecules from CO2 may open possibilities to use chemolithoautotrophic microorganisms for the sustainable production of important biomolecules.
... The use of mixed culture is a desirable solution for microalgae in order to dominate contamination risk and improve economic feasibility. Mooij et al. (2013) proposed the term "survival of the fattest", an approach for species enrichment with high storage productivity in the culture of mixed microalgae. Afterwards, a gravimetric enrichment technique could be used to screen lipid-and carbohydrate-accumulating species (Hassanpour et al. 2015). ...
Bioethanol is a fuel of plant origin obtained by the fermentation of simple sugars into alcohol. It can be used blended with fuels of fossil origin or almost pure in adapted vehicles (E-85, flex-fuel technology) particularly developed in Brazil. From a technological point of view, 3 generations coexist depending on the origin (from edible plants, lignocellulosic residues, or algae) of the sugars used for the fermentation. However, to date, the first generation still accounts for more than 95% of the 109 billion liters of ethanol available on the global market in 2019 despite many controversies about competition with foodstuffs and indirect changes in land use associated with the first generation. The obligation to reduce greenhouse gases emissions (GHG) to stay as close as possible to the 1.5 °C temperature raise objective by the end of the century that has been set in the 2015 Paris agreement, associated with the many changes in governments energy and mobility policies favoring electric vehicles in some parts of the world have led to a great uncertainty to foresee the future of this first-generation bioethanol technology.
... The use of mixed culture is a desirable solution for microalgae in order to dominate contamination risk and improve economic feasibility. Mooij et al. (2013) proposed the term "survival of the fattest", an approach for species enrichment with high storage productivity in the culture of mixed microalgae. Afterwards, a gravimetric enrichment technique could be used to screen lipid-and carbohydrate-accumulating species (Hassanpour et al. 2015). ...
Bioethanol consumption is projected to increase to 140 billion liters by 2029. However, climate change threatens the global production of the main bioethanol feedstocks, like sugarcane and corn. The search for new sources of biomass with greater resistance to these adverse conditions is essential to minimize impacts on biofuels production. In this context, two crops stand out: energy cane and Agave. Originally bred to improve the Saccharum genus feasibility to lignocellulosic bioethanol production, energy cane has physiological and biochemical characteristics that enhance robustness and could act as a direct substitute for sugarcane in traditional areas already suffering from irregular climate. On the other hand, Agave is a desert crop that has been domesticated as early as corn and has traditionally been used for alcoholic beverages and fibers but has never been applied for bioenergy purposes. With high water use efficiency, agaves can yield as much as sugarcane and can be used to slacken land competition and improve food and energy security. Both these crops present promising productivities and traits, such as high carbohydrates accumulation and drought resistance. However, they still face similar challenges to unravel its potential as new feedstocks for bioethanol in an Era of Climate Change.KeywordsEnergy caneAgaveBiofuelEthanolLignocellulosic
... The use of mixed culture is a desirable solution for microalgae in order to dominate contamination risk and improve economic feasibility. Mooij et al. (2013) proposed the term "survival of the fattest", an approach for species enrichment with high storage productivity in the culture of mixed microalgae. Afterwards, a gravimetric enrichment technique could be used to screen lipid-and carbohydrate-accumulating species (Hassanpour et al. 2015). ...
This chapter explores the main microbial breeding techniques for 1G and 2G bioethanol production, including classical genetic strategies (induced mutations, clonal selection, sexual hybridization, artificial hybridization, and evolutionary engineering) and those based on genetic transformation (deletion and regulation of genes, pooled-segregant whole genome sequence analysis, and CRISPR/CAS9 system). Saccharomyces cerevisiae has long been a popular model organism for breeding biological research; however, genetic manipulation of non-model microorganisms (e.g., Z. mobilis and Escherichia coli) has also been explored mainly for 2G ethanol production. Tools based on classical genetics are generally random and, therefore, less efficient. However, these techniques have the advantage of not needing prior knowledge about the gene of interest (facilitated procedure), and the microorganisms generated are not considered genetically modified. On the other hand, modifications based on genetic transformation result in more targeted improvements and overproduction of metabolites, although they are more expensive techniques and require extensive knowledge of intracellular biochemical pathways and regulatory mechanisms.
... Microalgae are unicellular, photosynthetic microorganisms with sizes in the range of 0.0002-0.002 mm and in filamentous forms with sizes of 0.1 mm or greater (Gerardo et al. 2015;Mooij et al. 2013; Barsanti and Gualtieri 2006). They are classified as red, green, brown, and blue-green algae; it is included in the Protista kingdom, not the Plant kingdom (Swapnil et al. 2014). ...
Technological improvements in dietary supplements and nutraceuticals have highlighted the significance of bioactive molecules in a healthy lifestyle. Eicosapentaenoic acid and Cervonic acid (DHA), omega-3 polyunsaturated fatty acids seem to be famed for their ability to prevent diverse physiological abnormalities. Selection of appropriate pretreatments and extraction techniques for extraction of lipids from robust microalgae cell wall are very important to retain their stability and bioactivity. Therefore, extraction techniques with optimized extraction parameters offer an excellent approach for obtaining quality oil with a high yield. Oils enriched in omega-3 are particularly imperiled to oxidation which ultimately affects customer acceptance. Bio active encapsulation could be one of the effective approaches to overcome this dilemma. This review paper aims to give insight into the cultivation methods, and downstream processes, various lipid extraction approaches, techniques for retaining oxidative stability, bioavailability and food applications based on extracted or encapsulated omega-3.
... Although many microbes have been enriched in a wide range of conditions, all of these enrichments are based on imposing a specific selective pressure in either a continuous or batch system. Recent work has demonstrated that through imposing dynamic process conditions an additional wealth of microbial diversity is revealed, compared to traditional chemostat or batch cultivation (6)(7)(8)(9). Essentially all natural environments are subject to ever changing conditions, from scarce nutrient availability to its sudden abundance and more regular day-night cycles leading to fluctuations in temperature and substrate supply. An abundant portion of microbes evolved under dynamic conditions and as such developed different functional strategies to exploit a competitive edge. ...
Determining the functional development and dominant competitive strategy in microbial community enrichments is complicated by the extensive measurement campaigns required for off-line system analysis. This study demonstrates that detailed system characterization of aerobic pulse fed enrichments can be established using on-line measurements combined with automated data analysis. By incorporating the physicochemical processes in on-line data processing with a Particle Filter and kinetic process model, an accurate reconstruction of the dominant biological rates can be made. We hereby can differentiate between storage compound production and biomass growth in sequencing batch bioreactors. The method proposed allows for close monitoring of changes in functional behavior of long-running enrichment cultures, without the need for off-line samples, therewith enabling the identification of new insights in process dynamics with a minimal experimental effort. Even though a specific example application of the method proposed is described here, the approach can readily be extended to a wide range of dynamic experimental systems that can be characterized based on on-line measurements.
... When the cells reached ≥ 3.0 g/L concentration, nitrogen starvation was implemented in an N-free BG-11 medium for 2 days. Nitrogen-starved microalgae can assimilate ammonia faster, and only the microalga assimilating ammonia the fastest survives in the mixed algal culture (Mooij et al., 2015). They were harvested by centrifugation at 1590 × g for 15 min (RC 5B plus, Thermo Fisher, USA). ...
Microalgal treatment of undiluted raw piggery wastewater is challenging due to ammonia toxicity and a deep dark color hampering photosynthesis. To overcome these problems, (1) a microalga (Coelastrella sp.) was isolated from an ammonia-rich environment, (2) the wastewater treatment was divided into two steps: a heterotrophic process followed by a mixotrophic process, and (3) a narrower transparent photobioreactor was employed with higher light intensity in the mixotrophic process. Coelastrella sp. removed 99% of ammonia, 92% of chemical oxygen demand (COD), and 100% of phosphorus during the 4-day process. Acetate in the wastewater relieved the ammonia stress on microalgae and promoted algal lipid and triacylglycerol productivity. Oxidative stability and low-temperature fluidity of triacylglycerols in lipids were improved by means of an altered fatty acid profile. Aside from the overall microalgal treatment performance, the proposed processing of piggery wastewater yielded a material suitable for possible production of algal biodiesel of better quality.
... In some phytoplankton-dominated lakes, cyanobacteria are harvested for food (e.g. Spirulina or Arthrospira; Habib, 2008), and phytoplankton-dominated lakes may provide a genetic resource for the synthesis of valuable biochemicals (Mooij, Stouten, Tamis, van Loosdrecht, & Kleerebezem, 2013;Muys et al., 2019). ...
• Shallow lakes can shift between stable states as a result of anthropogenic or natural drivers. Four common stable states differ in dominant groups of primary producers: submerged, floating, or emergent macrophytes or phytoplankton. Shifts in primary producer dominance affect key supporting, provisioning, regulating, and cultural ecosystem services supplied by lakes. However, links between states and services are often neglected or unknown in lake management, resulting in conflicts and additional costs.
• Here, we identify major shallow lake ecosystem services and their links to Sustainable Development Goals (SDGs), compare service provisioning among the four ecosystem states and discuss potential trade‐offs.
• We identified 39 ecosystem services potentially provided by shallow lakes. Submerged macrophytes facilitate most of the supporting (86%) and cultural (63%) services, emergent macrophytes facilitate most regulating services (60%), and both emergent and floating macrophytes facilitate most provisioning services (63%). Phytoplankton dominance supports fewer ecosystem services, and contributes most to provisioning services (42%).
• The shallow lake ecosystem services we identified could be linked to 10 different SDGs, notably zero hunger (SDG 2), clean water and sanitation (SDG 6), sustainable cities and communities (SDG 11), and climate action (SDG13).
• We highlighted several trade‐offs (1) among ecosystem services, (2) within ecosystem services, and (3) between ecosystem services across ecosystems. These trade‐offs can have significant ecological and economic consequences that may be prevented by early identification in water quality management.
• In conclusion, common stable states in shallow lakes provide a different and diverse set of ecosystem services with numerous links to the majority of SDGs. Conserving and restoring ecosystem states should account for potential trade‐offs between ecosystem services and preserving the natural value of shallow lakes.
... Such trait combinations are normally mutually exclusive. Approaches include genetic engineering (Zeng et al., 2011), directed evolution via successive rounds of mutagenesis and selection (Johnson et al., 2016;Lewin et al., 2016), and selection on the existing levels of genetic variation in a population (Mooij et al., 2013;Shurin et al., 2016). ...
Drawing insights from multiple disciplines is essential for finding integrative solutions that are required to tackle complex environmental problems. Human activities are causing unprecedented influence on global ecosystems, culminating in the loss of species and fundamental changes in the selective environments of organisms across the tree of life. Our collective understanding about biological evolution can help identify and mitigate many of the environmental problems in the Anthropocene. To this end, we propose a stronger integration of environmental sciences with evolutionary biology.
... Microalgae are considered as a lowcost novel expression system for the biosynthesis of biopolymers (Daniell et al., 2009;Hempel et al., 2011). It is known that microalgae under selective environment like absence of nutrients and varied photoperiod produce storage compounds which are natural polymers (Mooij et al., 2013). Microalgae excrete extracellular polymeric substance (EPS) into its environment comprising of polysaccharides, proteins, lipids, uronic and nucleic acids under controlled conditions. ...
Microalgae due to its metabolic versatility have received a focal attention in the biorefinery and bioeconomy context. Microalgae products have broad and promising application potential in the domain of renewable fuels/energy, nutraceutical, pharmaceuticals and cosmetics. Biorefining of microalgal biomass in a circular loop with an aim to maximize resource recovery is being considered as one of the sustainable option that will have both economical and environmental viability. The expansive scope of microalgae cultivation with self-sustainability approach was discussed in this communication in the framework of blue-bioeconomy. Microalgae based primary products, cultivation strategies, valorization of microalgae biomass for secondary products and integrated biorefinery models for the production of multi-based products were discussed. The need and prospect of self-sustainable models in closed loop format was also elaborated.
... Therefore, the ad-hoc control of microalgae biomass composition during the photosynthetic biogas upgrading can enhance the economic sustainability of the process by producing a biomass feedstock with a tailored valorization potential. In this context, Kleerebezem and co-workers reported a significant increase in the carbohydrate content of microalgae when nutrients were supplied during the dark period, which promoted the enrichment of microalgae capable of growing based on the intracellular polyglucose accumulated during the illuminated period in excess of CO 2 [16]. ...
The performance of a pilot high rate algal pond (HRAP) interconnected with a biogas absorption column during the simultaneous upgrading of biogas and treatment of digestate was evaluated under two innovative biogas and nutrient supply strategies. Process operation with biogas supply during the night at a liquid recirculation/biogas ratio of 0.5 to prevent N2 and O2 stripping resulted in a biomethane complying with most international regulations for injection into natural gas grids (99.1 ± 1% CH4, 0.5 ± 0.2% CO2, 0.6 ± 0.5% N2 and 0.07 ± 0.08% O2). The potential of this technology to remove methyl mercaptan (MeSH), toluene and hexane from biogas (typically present at trace levels) was assessed, for the first time, with removal efficiencies under steady-state correlating with pollutant hydrophobicity (7 ± 7% for hexane, 66 ± 4% for MeSH and 98 ± 1% for toluene). Finally, the supply of digestate during the dark period shifted both microalgae population structure and biomass composition in the HRAP without a significant impact on biomethane quality. Overall, the removal of nitrogen and phosphorous from digestate in the HRAP was almost complete (96–99%) regardless of the nutrient supply strategy.
... Furthermore, higher lipid accumulation in Pi-Vitamin medium may also lead to conclude that the supplementation provides a selective environment in which species with higher lipid production capacity are enriched. There are other studies with the aim of selective environment for lipid-producing algae (Hassanpour et al., 2015;Mooij et al., 2013). ...
The present study investigated the interaction between starch and lipid accumulation was in a green microalgae enrichment culture. The objective was to optimize the lipid content by manipulation of the medium in regular batch culture. Two medium designs were evaluated: first a high ortho-P concentration with vitamin supplement (Pi-vitamins supplemented medium), second normal growth medium (control). Both media contained a low amount of nitrogen which was consumed during batch growth in three days. The batch experiments were prolonged continued for another four days with the absence of soluble nitrogen in the medium. When the mixed microalgal culture was incubated with in the Pi-vitamin supplemented medium, the lipid and starch content of the culture increased within the first three days to 102.0 ± 5.2 mg.l⁻¹ (12.7 ± 0.6% of DW) and 31.7 ± 1.6 mg.l⁻¹ (4.0 ± 0.2% of DW), respectively. On the last day of the experiment, the lipid and starch content in Pi-vitamin medium increased to 663.1 ± 32.5 (33.4 ± 1.6% of DW) and 127.5 ± 5.2 (6.4 ± 0.3% of DW) mg.l⁻¹. and However, the lipid and starch content in the control process, reached to 334.7 ± 16.4 (20.1 ± 1.0% of DW) and 94.3 ± 4.6 (5.7 ± 0.3% of DW), respectively. The high Pi-vitamin medium induced storing lipid formation clearly while the starch formation was not affected. The lipid contents reported here are among the high reported in the literature, note that already under full growth conditions significant lipid levels occurred in the algal enrichment culture. The high lipid productivity of the reported mixed microalgae culture provides an efficient route for efficient algal biodiesel production. This article is protected by copyright. All rights reserved
... However, this new combination is still under investigation and a hybrid system can be developed: While microalgae remove the nutrient, they serve as a substrate for anaerobic digestion to produce methane and to recover nutrients. In the literature, there are limited studies on this (Alcántara et al. 2013;Mahdy et al. 2014;Mooij et al. 2013;Posadas et al. 2013). These studies showed that algal biomass is a potential co-substrate for biogas production. ...
... Particularly, no study on ANN modeling of enzymatic hydrolysis of pure or mixed microalgae culture has yet been reported. Application of mixed culture of microalgae will decline the risk of system contamination, and thus operation in different conditions and process feasibility will be increased (Hassanpour et al., 2015;Mooij et al., 2013). ...
The efficient production of reducing sugars is an extremely important requirement in the utilization of microalgae as a feedstock in bioethanol production. In this study, for the first time, the time course of reducing sugar production during starch hydrolysis of mixed microalgal biomass under different operational conditions was modeled by two different methods; a-Michaelis-Menten kinetic model and b-artificial neural network (ANN) method. The results from both models revealed that predicted values are in good agreement with the experimental results. Also, sensitivity analysis indicated that the kinetic model results are less sensitive to Km and Ki than to . The applied ANN was a feed-forward back propagation network with Levenberg-Marquardt algorithm. It was found that the order of relative importance of the input variables on reducing sugar concentration predicted by ANN model was as follows: pH>substrate concentration>temperature>hydrolysis time. Subsequently, the results indicated that the maximum reducing sugar yield (96.3%) was achieved by adding enzymes with the sequence of first cellulases, at 50 °C, pH 4.5, second α -amylase, at 70 °C, and pH 6 with a substrate concentration of 50 g/L. These findings may be useful for improving the enzymatic hydrolysis of mixed microalgae for bioethanol production.
... reducing the carbon flow towards polysaccharide and glycoprotein matrix of the cell wall). Besides metabolic engineering, also adaptive laboratory evolution to a selective pressure [48] combined with FACS (Fluorescence Activated Cell Sorting) [38,49,50] or RACS (Raman Activated Cell Sorting) [51] can lead to increased TAG productivities. ...
Background
Microalgal triglycerides (TAGs) hold great promise as sustainable feedstock for commodity industries. However, to determine research priorities and support business decisions, solid techno-economic studies are essential. Here, we present a techno-economic analysis of two-step TAG production (growth reactors are operated in continuous mode such that multiple batch-operated stress reactors are inoculated and harvested sequentially) for a 100-ha plant in southern Spain using vertically stacked tubular photobioreactors. The base case is established with outdoor pilot-scale data and based on current process technology.
Results
For the base case, production costs of 6.7 € per kg of biomass containing 24% TAG (w/w) were found. Several scenarios with reduced production costs were then presented based on the latest biological and technological advances. For instance, much effort should focus on increasing the photosynthetic efficiency during the stress and growth phases, as this is the most influential parameter on production costs (30 and 14% cost reduction from base case). Next, biological and technological solutions should be implemented for a reduction in cooling requirements (10 and 4.5% cost reduction from base case when active cooling is avoided and cooling setpoint is increased, respectively). When implementing all the suggested improvements, production costs can be decreased to 3.3 € per kg of biomass containing 60% TAG (w/w) within the next 8 years.
Conclusions
With our techno-economic analysis, we indicated a roadmap for a substantial cost reduction. However, microalgal TAGs are not yet cost efficient when compared to their present market value. Cost-competiveness strictly relies on the valorization of the whole biomass components and on cheaper PBR designs (e.g. plastic film flat panels). In particular, further research should focus on the development and commercialization of PBRs where active cooling is avoided and stable operating temperatures are maintained by the water basin in which the reactor is placed.
Electronic supplementary material
The online version of this article (doi:10.1186/s13068-017-0873-2) contains supplementary material, which is available to authorized users.
... Thus, mixed cultures are considered as an attractive research area compared to traditional pure culture. Mooij et al. introduced the concept of ''survival of the fattest", a strategy for enrichment of species with a high storage compound productivity in mixed microalgae culture [18]. Later on, Hassanpour et al. applied a gravimetric enrichment method for screening carbohydrate and lipid accumulating species in a mixed microalgae culture [19]. ...
Mixed microalgae cultures are considered as an attractive research area compared to traditional pure culture to dominate cultivation contamination risk and enhance economic feasibility of large-scale biofuel production. However, pre-treatment and bioethanol production from mixed microalgae culture has not been reported yet. Therefore, this study was aimed to evaluate the effect of different pre-treatment strategies including acidic, alkaline, and enzymatic hydrolysis on the sugar extraction from mixed microalgae. Besides, the effects of MgSO4 and CaCl2 as lewis acids in acidic pre-treatment on reducing sugar yield were studied.
... Open pond systems offer advantages compared to closed photobioreactor systems in terms of energy input and ease of operation. Recent work by Mooij et al. (2013) showed that one of the main disadvantages of open pond systems, contamination by less productive species, can be overcome by creating a selective environment. The temperature for the culture was assumed to be optimal and the energy-input of the microalgal cultivation was assumed to be covered by renewable resources, such as wind-or tidal power. ...
The disruption of nutrient cycles caused by human activities such as agriculture and burning fossil fuels is impacting ecosystem services on global and local scales. The increasing concentration of carbon dioxide in the atmosphere contributes to rising global temperatures and ocean acidification, whereas the accumulation of nutrients in water systems is leading to degradation of water quality and biodiversity. City populations play a major role in carbon dioxide and nutrient emissions as ‘end consumers’ of resources. The current challenge towards more resource-efficient cities is to transform urban metabolism from linear to cyclical. Discharged nutrients and carbon dioxide can be used as input for algae, which fixate carbon very efficiently into energetic storage compounds as starch or lipids. However, cities often lack the space to implement large-scale algae production. This article evaluates the potential of reusing nutrients and carbon dioxide to produce algae, food and biofuel on water nearby coastal and delta cities. First, nutrients and carbon dioxide discharge is estimated and two scenarios are developed. From the cities nutrient production, the potential algal yield is evaluated and translated into feed, food and oil yields. Two delta cities are chosen as case studies: Rotterdam and Metro Manila. The conclusion of this article is that Floating Production can help cities increasing their resilience in the field of food and energy. Floating Production can also contribute to a solution for global land shortage. The combination of food and energy production with floating urban development provides a climate-proof urban expansion in delta and coastal areas.
... . Indeed, data from this experimentation are in the low range, and this is in agreement with the low lipid content of the algal biomass. Indeed, the algal BMP is expected to be higher for those microalgae that are naturally richer in lipid or that are grown under environmental conditions that con induce lipid production (among others Li et al. 2008;Mooij et al. 2013). ...
This paper discusses the possibility of including the culturing of microalgae within a conventional wastewater treatment sequence by growing them on the blackwater (BW) from biosolid dewatering to produce biomass to feed the anaerobic digester. Two photobioreactors were used: a 12 L plexiglas column for in-door, lab-scale tests and a 85 L plexiglas column for outdoor culturing. Microalgae (Chlorella sp and Scenedesmus sp) could easily grow on the tested blackwater. The average specific growth rate in indoor and outdoor batch tests was satisfactory, ranging between 0.14-0.16 d-1. During a continuous test performed under outdoor conditions from May to November, in which the off-gas from the combined heat and power unit was used as the CO2 source, an average biomass production of 50 mgTSS L-1 d-1 was obtained. However, statistical analyses confirmed that microalgal growth was affected by environmental conditions (temperature and season) and that it was negatively correlated with the occurrence of nitrification.
Finally, the biochemical methane potential of the algal biomass was slightly higher than that from waste sludge (208 mLCH4 gVS-1 vs. 190 mLCH4 gVS-1).
... Recently, Mooij et al. (2015) have proposed to apply selection pressures for microalgae culture in order to select species of interest, and to avoid contaminations. In particular, periodic nutrient stress can be carried out for the selection of microalgae with a high storage capacity (Masci et al., 2008;Mooij et al., 2013). ...
Microalgae have recently attracted attention for their potential to produce high added compounds, proteins, and even biofuels. Our paper seeks to develop a control strategy for light-limited continuous culture imposing a stress for which microalgae have to adapt. This operating mode - called photoinhibistat - consists, for a culture with a constant dilution rate, in varying the incident light in order to regulate the light at the bottom of the reactor, inducing a light stress. Based on a simple model of light-limited growth, we analyze the dynamics of the photoinhibistat in monoculture and in competition. It appears that the photoinhibistat can be used to select, from the initial microalgae population, the strain with the highest resistance to photoinhibition.
... This has been supported by the observation that polycultures are more stable and productive tan monocultures under some scenarios of growth (Shurin et al., 2013). Furthermore, it has been shown that it is possible to use an environmental selective pressure to force consortia of microalgae to exhibit a certain train, such as increased lipid productivity (Mooij et al., 2013). These methods are attractive because they bypass chemical use and harness the dynamics of natural communities (Smith and Mcbride, 2015;Smith et al., 2010). ...
Microalgal cultivation that takes advantage of solar energy is one of the most cost-effective systems for the biotechnological production of biofuels, and a range of high value products, including pharmaceuticals, fertilizers and feed. However, one of the main constraints for the cultivation of microalgae is the potential contamination with biological pollutants, such as bacteria, fungi, zooplankton or other undesirable microalgae. In closed bioreactors the control of contamination requires the sterilization of the media, containers, and all materials, which increases the cost of production, whereas in open pond systems severely limits the number of species that can be cultivated. Here, we report the metabolic engineering of Chlamydomonas reinhardtii to use phosphite as its sole phosphorus source by expressing the ptxD gene from Pseudomonas stutzeri WM88, which encodes a phosphite oxidoreductase able to oxidize phosphite into phosphate using NAD as a cofactor. Engineered C. reinhardtii lines are capable of becoming the dominant species in a mixed culture when fertilized with phosphite as a sole phosphorus source. Our results represent a new platform for the production of microalgae, potentially useful for both closed photobioreactors and open pond systems without the need for using sterile conditions nor antibiotics or herbicides to prevent contamination with biological pollutants. This article is protected by copyright. All rights reserved.
Microalgae are photosynthetic, unicellular microorganisms also known as phytoplankton. They are small plant-like entities. In this research, the sample were collected from cement factory in a sterilised 20L container wrapped with foil paper and were transported down to Federal University, Wukari where it was kept in refrigerator at biochemistry laboratory. Blue-Green media (BG-11) was prepared. Wastewater containing Microalgae obtained from cement wastewater pond were cultivated in BG-11 medium to determine the growth of the organism. BG-11 medium contained macronutrients, trace metals and some vitamins which aid the growth of the organism. The wastewater sample containing with suspected microalgae was inoculated (inoculum ratio = 25%) and incubated under atmospheric CO2 at room temperature (30±2°C) in our laboratory for two to three weeks during the incubation period. In order to purify the isolates, the upper growth layer was first decanted into a freshly produced medium and then plated on BG-11 media that had been solidified with 1% agar-agar. For several of the cultures, growth on the agar plates continued for around three weeks. Following repeated sub-culturing, the emerging greenish colonies were re-emerged into a sterile BG-11 agar medium. In isolation of organism from the industrial cement waste water, the isolate was identified by morphological and molecular identification by extracting the DNA, run the electrophoretic analysis and PCR using primers for 18S rRNA eukaryotic microalgal and then run the sequence analysis. The results of this study obtained, indicated that, the electrophoretic result show the band has 1800-2000base pair and the organism isolated from the industrial cement waste water were chlorogonium sp. with a percent similarity of 78.65% and accession number of OR886595 based on data Gene Bank blast results.
First-generation bioethanol, made from edible feedstocks, are currently not regarded as a sustainable source due to the food versus fuel dilemma. Second-generation bioethanol, despite being made from non-edible sources, are not cost-effective owing to their high production cost. To avoid the drawbacks of its predecessor, third-generation bioethanol from microalgae and macroalgae have been considered a promising replacement to depleting petroleum fuels. In this chapter, the progress of research on micro and macroalgae for third-generation bioethanol production is discussed. This chapter thoroughly explains the use of microalgae and macroalgae for bioethanol production, starting from strains selection, cultivation, harvesting and drying, to hydrolysis, fermentation and distillation. To become a competitive source for bioethanol, the production of microalgae should be cheap and highly efficient. Therefore, each of the above processes should be improved and optimised. At the end of this chapter, the future direction on bioethanol production from microalgae and macroalgae is highlighted.
The enzymatic hydrolysis of microalgae has already been studied for bioethanol production. However, the enzymatic hydrolysis of microalgae under various fluid hydrodynamic conditions has not been simulated yet. Accordingly, the present study investigated the effect of various stirrer speed values, impeller types, and the presence of baffles on glucose extraction from the mixed microalgae using cellulase. The Michaelis-Menten kinetic constants of enzymatic hydrolysis were calculated in the AQUASIM and were used for simulation in COMSOL. The simulated values agreed with the experimental results and revealed that the low stirring speed reduced glucose extraction through undesirable enzyme distribution and the formation of regions with a high concentration of hydrolysis products to inhibit the enzyme's operation. The higher uniformity of glucose concentration at higher stirring speeds compared to lower stirring speeds ensured the higher efficiency of the mixing process in this reactor. The study findings suggested that a sufficient understanding of the mixing effects in the enzymatic hydrolysis could improve the economic feasibility of the process. Furthermore, compared to the unbaffled reactor, the proper mixing in the baffled reactor increased the enzymatic hydrolysis rate. It was found that altering the impeller type leads to a negligible change in the enzymatic hydrolysis rate.
Beta-carotene, a pigment found in plants, is mainly produced by microalgae. Nevertheless, this production has only been investigated in pure cultures. Beta-carotene production through mixed culture eliminates the costly procedure of sterilization and contamination prevention needed for pure cultures. In this study, for the first time, the growth, beta-carotene, and chlorophyll production of a mixed culture of microalgae from Caspian Sea was investiagted under different stress conditions. At the condition of tripled light intensity and nitrogen starvation, beta-carotene content increased from 18.03 to 43.8 and 46.5 mol beta-carotene g ⁻¹ protein, respectively. However, the salinity of 4 mol L ⁻¹ caused the beta-carotene content to fall to zero. The blank sample reached a constant value of 23 mol beta-carotene g ⁻¹ protein. The comparable results with the specific monoculture species exhibit the high potential of a mixed culture of microalgae for beta-carotene production without need of the high sterilization cost. Nevertheless, more research is needed for where it can be a good substitute for pure culture.
In this work, the algal biomass productivity and its lipid content were explored using a database containing 4670 instances extracted from the experimental results reported in 102 published articles. First, the influences of critical factors such as microalgae species, cultivation conditions, light intensity, CO2 amount, nutrient concentrations, reactor type, stress conditions, cell disruption methods, and lipid extraction solvents on the biomass and lipid production were reviewed. Then, the database was analyzed using machine learning techniques; decision trees were utilized to determine the combination of variables leading to high biomass and lipid content while association rule mining was used to find the specific conditions leading to very high biomass and lipid levels. Decision tree analysis discovered 11 different combinations of variables leading to high biomass productivity and 13 combinations for high lipid content; whereas, association rule mining analysis helped to identify the levels of specific factors for very high biomass and lipid production. It was then concluded that machine learning methods can help to determine the best conditions for optimum biomass growth and lipid yield for microalgae to manufacture renewable biofuels, and this can guide the planning of new experimental works.
A high ammonia concentration and chemical oxygen demand (COD) in piggery wastewater force it to be diluted before conventional microalgal treatment to reduce ammonia toxicity. Incomplete treatment of ammonia and COD in piggery wastewater may cause eutrophication, resulting in algal blooms. This study tried to treat raw piggery wastewater without dilution, using three strains of microalgae (Chlorella sorokiniana, Coelastrella sp. and Acutodesmus nygaardii) that outcompeted other algae under heterotrophic, mixotrophic, and autotrophic conditions, respectively, through adaptive evolution at high ammonia concentration. The three stepwise processes were designed to remove (1) small particles, COD, and phosphorus in the 1st heterotrophic C. sorokiniana cultivation, (2) ammonia and COD in the 2nd mixotrophic Coelastrella sp. cultivation, and (3) the remaining ammonia in the 3rd photoautotrophic A. nygaardii cultivation. To enhance ammonia uptake rate, each algal species were inoculated after 2-day nitrogen starvation. When the N-starved three species were inoculated at each step sequentially at 7 g/L for 2 days, the final phosphorus, COD, and ammonia removal efficiencies were 100% (16.4 to 0 mg/L), 92% (6820 to 545 mg/L), 90% (850 to 81 mg/L) and turbidity (99%) after total 6 days.
Use of diverse algal polycultures could enhance the viability of mass algal cultivation by increasing the productivity and stability of production. However, there are multiple approaches for selecting productive polycultures, ranging from a synthetic ecology approach using carefully selected laboratory strains, to collection and cultivation of naturally occurring polycultures. In this study, we compared the performance of a systematically assembled and a naturally occurring polyculture under two conditions: 1) pilot-scale, semi-continuous culture conditions and 2) grazing and resource stress conditions. The naturally occurring polyculture had 25% higher mean biomass productivity and 19% higher N-removal efficiency than the assembled polyculture under semi-continuous culture. Differences became even more pronounced under grazing and resource stress conditions; mean biomass productivity was 60% higher in the naturally occurring polyculture when exposed to stress. Lipid and protein content (% dry mass) were significantly higher in the assembled polyculture, while carbohydrate content was higher in the naturally occurring polyculture. The naturally occurring polyculture maintained higher diversity than the assembled polyculture throughout the experiment, suggesting a greater degree of niche complementarity as a potential mechanism for differences in biomass yields and nutrient uptake. Our results show that systematically engineered algal communities may not always lead to greater productivity than algal communities found in nature per se. We suggest that future studies using polycultures assembled from lab strains should test them against those occurring naturally; collectively, this would both serve as a benchmark for progress in synthetic ecology and would test whether there are generalities in the performance of natural vs. assembled polycultures.
The prospect of using constructed communities of microalgae in algal cultivation was confirmed in this study. Three different algal communities, constructed of diatoms (Diatom), green algae (Green) and cyanobacteria (Cyano), each mixed with a natural community of microalgae were cultivated in batch and semi‐continuous mode and fed CO2 or cement flue gas (12‐15 % CO2). Diatom had the highest growth rate but Green had the highest yield. Changes in the community composition occurred throughout the experiment. Green algae were the most competitive group, while filamentous cyanobacteria were outcompeted. Euglenoids, recruited from scarce species in the natural community became a large part of the biomass in semi‐steady state in all communities. High temporal and yield stability were demonstrated in all communities during semi‐steady state. Valuable products (lipids, proteins and carbohydrates) comprised of 61.5 ±5 % of ash free biomass and were similar for the three communities with lipids ranging 14–26 % of dry mass (DM), proteins (15‐28 % DM) and carbohydrates (9‐23 % DM). Our results indicate that culture functions (stability, biomass quality) were maintained while dynamic changes occurred in community composition. We propose that a multi‐species community approach can aid sustainability in microalgal cultivation, through complementary use of resources and higher culture stability.
This article is protected by copyright. All rights reserved
A paradigm shift is underway in wastewater treatment as the industry heads toward ~3% of global electricity consumption and contributes ~1.6% of greenhouse gas emissions. Although incremental improvements to energy efficiency and renewable energy recovery are underway, studies considering wastewater for carbon capture and utilization are few. This Review summarizes alternative wastewater treatment pathways capable of simultaneous CO2 capture and utilization, and demonstrates the environmental and economic benefits of microbial electrochemical and phototrophic processes. Preliminary estimates demonstrate that re-envisioning wastewater treatment may entirely offset the industry’s greenhouse gas footprint and make it a globally significant contributor of negative carbon emissions.
The capacity of microalgae to advance the limit of technology of nutrient recovery and accumulate storage carbon make them promising candidates for wastewater treatment. However, the extent to which these capabilities are influenced by microbial community composition remains poorly understood. To address this knowledge gap, 3 mixed phototrophic communities sourced from distinct latitudes within the continental United States (28° N, Tampa, FL; 36° N, Durham, NC; and 40° N, Urbana, IL) were operated in sequencing batch reactors (8 day solids residence time, SRT) subjected to identical diel light cycles with media addition at the start of the nighttime period. Despite persistent differences in community structure as determined via 18S rRNA (V4 and V8-V9 hypervariable regions) and 16S rRNA (V1-V3) gene amplicon sequencing, reactors achieved similar and stable nutrient recovery after 2 months (8 SRTs) of operation. Intrinsic carbohydrate and lipid storage capacity and maximum specific carbon storage rates differed significantly across communities despite consistent levels of observed carbon storage across reactors. This work supports the assertion that distinct algal communities cultivated under a common selective environment can achieve consistent performance while maintaining independent community structures and intrinsic carbon storage capabilities, providing further motivation for the development of engineered phototrophic processes for wastewater management.
Suspended growth, mixed community phototrophic wastewater treatment systems (including high-rate algal ponds and photobioreactors) have the potential to achieve biological nitrogen and phosphorus recovery with effluent nutrient concentrations below the current limit-of-technology. In order to achieve reliable and predictive performance, it is necessary to establish a thorough understanding of how design and operational decisions influence the complex community structure governing nutrient recovery in these systems. Solids residence time (SRT), a critical operational parameter governing growth rate, was leveraged as a selective pressure to shape microbial community structure in laboratory-scale photobioreactors fed secondary effluent from a local wastewater treatment plant. In order to decouple the effects of SRT and hydraulic retention time (HRT), nutrient loading was fixed across all experimental conditions and the effect of changing SRT on microbial community structure, diversity, and stability, as well as its impact on nutrient recovery, was characterized. Reactors were operated at distinct SRTs (5, 10, and 15 days) with diurnal lighting over long-term operation (>6 SRTs), and in-depth examination of the eukaryotic and bacterial community structure was performed using amplicon-based sequencing of the 18S and 16S rRNA genes, respectively. In order to better represent the microalgal community structure, this study leveraged improved 18S rRNA gene primers that have been shown to provide a more accurate representation of the wastewater process-relevant algal community members. Long-term operation resulted in distinct eukaryotic communities across SRTs, independent of the relative abundance of Operational Taxonomic Units (OTUs) in the inoculum. The longest SRT (15 days, SRT 15) resulted in a more stable algal community along with stable bacterial nitrification, while the shortest SRT (5 days, SRT 5) resulted in a less stable, more dynamic community. Although SRT was not strongly associated with overall bacterial diversity, the eukaryotic community of SRT 15 was significantly less diverse and less even than SRT 5, with a few dominant OTUs making up a majority of the eukaryotic community structure in the former. Overall, although longer SRTs promote stable bacterial nitrification, short SRTs promote higher eukaryotic diversity, increased functional stability, and better total N removal via biomass assimilation. These results indicate that SRT may be a key factor in not only controlling microalgal community membership, but community diversity and functional stability as well. Ultimately, the efficacy and reliability of NH4⁺ removal may be in tension with TN removal in mixed phototrophic systems given that lower SRTs may achieve better total N removal (via biomass assimilation) through increased eukaryotic diversity, biomass productivity, and functional stability.
Microalgae offer a multiplicity of applications for the production of bio-sourced compounds such as proteins, pigments, sugars and oils. However, the energy spent for algae culture and lipid extraction hinder the energetic viability of the process for the production of biofuel derived from algae oils. Among possible improvements, pulsed electric fields (PEF) may be used as a pre-treatment to extract valuable compounds from microalgae and making the process less energy demanding.This project started with a collaboration between the team of bio-micro-systems Biomis, laboratory SATIE, with the team of bio-process engineering laboratory LGPM to study in situ the effects of PEF on microalgae.First, a energetic study is performed in a micro-system specially built for this project to characterize in situ, the effect of various treatment parameters (pulse duration / electric field) on Chlamydomonas reinhardtii cells with high lipid content.Among the outputs of this study, an energetic optimization of PEF conditions shows that a high level of permeability and low energy consumption are obtained when using short pulses of 5 µs. Associated with an electric field of 4.5 kV/cm, the pores are reversible (80% of the cells) during few seconds, and with a field of 7 kV/cm or higher, the permeabilization is irreversible. Afterwards, this PEFpre-treatment is associated with solvent mixing (hexane) to evaluate if lipid extraction is improved.
A two-stage cultivation strategy was applied to mixed microalgae, which were first cultured in complete nutrient medium then switched to different nutrient-free mediums in order to assess the impact of nutrient starvation on intracellular biochemical components of mixed microalgae. The effects of nitrogen, sulfur and phosphorus starvation on cell counts, chlorophyll, carotenoid, protein, starch and lipid content of the mixed microalgae are compared in this study. The obtained results revealed that starch, as a dominant storage compound, was the highest in the nitrogen-free medium up to 49% of dry weight (DW). Protein and chlorophyll content declined slightly from 512 to 472.96 mg.l⁻¹ and 29.43 to 26.58 mg.l⁻¹ only in the case of nitrogen starvation. Nitrogen starvation showed the best result as it ceased cell division immediately whereby in the case of sulfur and phosphorus starvation, cell division was not interrupted as microalgae are a pool of phosphorus and store sulfur. Calculation of starch and lipid energy content on the basis of electron equivalent and J/mg dry weight showed the higher energy content of lipid in compare with that of starch for all nutrients starvation. Nitrogen deprivation condition represents the superlative energy value of storage compounds content with the value of 17.61 J/mg dry weight. The finding proves the potential of attractive and economically feasible mixed microalgae cultivation for high percentages of storage compounds production under nitrogen starvation
Microalgal treatment systems could advance nutrient recovery from wastewater by achieving effluent nitrogen (N) and phosphorus (P) levels below the current limit of technology, but their successful implementation requires an understanding of how design decisions influence nutrient uptake over daily (i.e., diel) cycles. This work demonstrates the ability to influence microalgal N:P recovery ratio via solids residence time (SRT) while maintaining complete nutrient removal across day/night cycles through carbon storage and mobilization. Experiments were conducted with two microalgal species, Scenedesmus obliquus and Chlamydomonas reinhardtii, in photobioreactors (PBRs) operated as cyclostats (chemostats subjected to simulated natural light cycles) with retention times of 6–22 days (S. obliquus) and 7–13 days (C. reinhardtii). Nutrient loading and all other factors were fixed across all experiments. Elevated SRTs (>8 days) achieved limiting nutrient concentrations (either N or P) below the detection limit throughout the diel cycle. N:P mass ratio in algal biomass was linearly correlated with SRT, varying from 9.9:1 to 5.0:1 (S. obliquus) and 4.7:1 to 4.3:1 (C. reinhardtii). Carbohydrate content of biomass increased in high irradiance and decreased in low irradiance and darkness across all experiments, whereas lipid dynamics were minimal over 24-h cycles. Across all nutrient-limited cultures, specific (i.e., protein-normalized) dynamic carbohydrate generally decreased with increasing SRT. Nighttime consumption of stored carbohydrate fueled uptake of nutrients, enabling complete nutrient limitation throughout the night. Dynamic carbohydrate consumption for nutrient assimilation was consistent with dark protein synthesis but less than that of heterotrophic growth, underscoring the need for algal process models to decouple growth from nutrient uptake in periods of low/no light. The ability to tailor microalgal N:P uptake ratio and target an optimal energy storage metabolism with traditional engineering process controls (such as SRT) may enable advanced nutrient recovery facilities to target continuous and reliable dual limitation of nitrogen and phosphorus.
Modern society is fueled by fossil energy produced millions of years ago by photosynthetic organisms. Cultivating contemporary photosynthetic producers to generate energy and capture carbon from the atmosphere is one potential approach to sustaining society without disrupting the climate. Algae, photosynthetic aquatic microorganisms, are the fastest growing primary producers in the world and can therefore produce more energy with less land, water, and nutrients than terrestrial plant crops. We review recent progress and challenges in developing bioenergy technology based on algae. A variety of high-value products in addition to biofuels can be harvested from algal biomass, and these may be key to developing algal biotechnology and realizing the commercial potential of these organisms. Aspects of algal biology that differentiate them from plants demand an integrative approach based on genetics, cell biology, ecology, and evolution. We call for a systems approach to research on algal biotechnology rooted in understanding their biology, from the level of genes to ecosystems, and integrating perspectives from physical, chemical, and social sciences to solve one of the most critical outstanding technological problems.
This paper presents a continuous-flow cell screening device to isolate and separate microalgae cells (Chlamydomonas reinhardtii) based on lipid content using high frequency (50 MHz) dielectrophoresis. This device enables screening of microalgae due to the balance between lateral DEP forces relative to hydrodynamic forces. Positive DEP force along with amplitude-modulated electric field exerted on the cells flowing over the planar interdigitated electrodes, manipulated low-lipid cell trajectories in a zigzag pattern. Theoretical modelling confirmed cell trajectories during sorting. Separation quantification and sensitivity analysis were conducted with time-course experiments and collected samples were analysed by flow cytometry. Experimental testing with nitrogen starved dw15-1 (high-lipid, HL) and pgd1 mutant (low-lipid, LL) strains were carried out at different time periods, and clear separation of the two populations was achieved. Experimental results demonstrated that three populations were produced during nitrogen starvation: HL, LL and low-chlorophyll (LC) populations. Presence of the LC population can affect the binary separation performance. The continuous-flow micro-separator can separate 74% of the HL and 75% of the LL out of the starting sample using a 50 MHz, 30 voltages peak-to-peak AC electric field at Day 6 of the nitrogen starvation. The separation occurred between LL (low-lipid: 86.1% at Outlet # 1) and LC (88.8% at Outlet # 2) at Day 9 of the nitrogen starvation. This device can be used for onsite monitoring; therefore, it has the potential to reduce biofuel production costs.
Although open outdoor pond systems are the most economically viable option for mass cultivation of algae as a biofuel source, such systems face a number of limitations. Open ponds experience environmental fluctuations (i.e., light levels, nutrient ratios, and temperature), invasion pressure by undesired algal species, pathogen infections, and herbivory by invading zooplankton, all of which may negatively influence the system's overall harvestable yield. Using ecological principles to address the limitations of open-pond cultivation is a promising direction in algal biofuel research. This review surveys the growing body of work on these topics and offers a mechanistic framework for optimizing algal biofuel production while minimizing the negative effects of invasion, infection, and herbivory. High levels of productivity (in terms of biomass and lipids) are crucial for viable biofuel production and can be achieved by increasing algal diversity and assembling communities based on species' eco-physiological traits. Herbivory can be significantly reduced by choosing algal species resistant to grazing or by introducing biotic controls on herbivores. Diverse assemblages of algal species can be constructed to fill in the available ecological niche space, leading not only to high productivity but also reduced invasibility by undesired strains and potentially reduced susceptibility to algal diseases. Optimization of the mass cultivation of algae requires an interdisciplinary approach that includes using ecological principles for designing productive, resistant, and resilient algal communities.
Background
Microalgae are a promising platform for producing neutral lipids, to be used in the application for biofuels or commodities in the feed and food industry. A very promising candidate is the oleaginous green microalga Scenedesmus obliquus, because it accumulates up to 45% w/w triacylglycerol (TAG) under nitrogen starvation. Under these conditions, starch is accumulated as well. Starch can amount up to 38% w/w under nitrogen starvation, which is a substantial part of the total carbon captured. When aiming for optimized TAG production, blocking the formation of starch could potentially increase carbon allocation towards TAG. In an attempt to increase TAG content, productivity and yield, starchless mutants of this high potential strain were generated using UV mutagenesis. Previous studies in Chlamydomonas reinhardtii have shown that blocking the starch synthesis yields higher TAG contents, although these TAG contents do not surpass those of oleaginous microalgae yet. So far no starchless mutants in oleaginous green microalgae have been isolated that result in higher TAG productivities.
Results
Five starchless mutants have been isolated successfully from over 3,500 mutants. The effect of the mutation on biomass and total fatty acid (TFA) and TAG productivity under nitrogen-replete and nitrogen-depleted conditions was studied. All five starchless mutants showed a decreased or completely absent starch content. In parallel, an increased TAG accumulation rate was observed for the starchless mutants and no substantial decrease in biomass productivity was perceived. The most promising mutant showed an increase in TFA productivity of 41% at 4 days after nitrogen depletion, reached a TAG content of 49.4% (% of dry weight) and had no substantial change in biomass productivity compared to the wild type.
Conclusions
The improved S. obliquus TAG production strains are the first starchless mutants in an oleaginous green microalga that show enhanced TAG content under photoautotrophic conditions. These results can pave the way towards a more feasible microalgae-driven TAG production platform.
Significance
As global CO 2 levels rise and fossil fuel abundance decreases, the development of alternative fuels becomes increasingly imperative. Biologically derived fuels, and specifically those from microalgae, are promising sources, but improvements throughout the production process are required to reduce cost. Increasing lipid yields in microalgae without compromising growth has great potential to improve economic feasibility. We report that disrupting lipid catabolism is a practical approach to increase lipid yields in microalgae without affecting growth or biomass. We developed transgenic strains through targeted metabolic engineering that show increased lipid accumulation, biomass, and lipid yields. The target enzyme’s ubiquity suggests that this approach can be applied broadly to improve the economic feasibility of algal biofuels in other groups of microalgae.
Microalgae represent one of the most promising groups of candidate organisms for replacing fossil fuels with contemporary primary production as a renewable source of energy. Algae can produce many times more biomass per unit area than terrestrial crop plants, easing the competing demands for land with food crops and native ecosystems. However, several aspects of algal biology present unique challenges to the industrial-scale aquaculture of photosynthetic microorganisms. These include high susceptibility to invading aquatic consumers and weeds, as well as prodigious requirements for nutrients that may compete with the fertiliser demands of other crops. Most research on algal biofuel technologies approaches these problems from a cellular or genetic perspective, attempting either to engineer or select algal strains with particular traits. However, inherent functional trade-offs may limit the capacity of genetic selection or synthetic biology to simultaneously optimise multiple functional traits for biofuel productivity and resilience. We argue that a community engineering approach that manages microalgal diversity, species composition and environmental conditions may lead to more robust and productive biofuel ecosystems. We review evidence for trade-offs, challenges and opportunities in algal biofuel cultivation with a goal of guiding research towards intensifying bioenergy production using established principles of community and ecosystem ecology.
The goal of this study was to investigate the time response of two major carbon (C) reserves, respectively neutral lipids (NL) and total carbohydrate (TC), in the Haptophyte Isochrysis sp. growing in nitrogen (N)-sufficient or N-starved conditions and under light:dark (L:D) cycles. Experiments were carried out in a cyclostat culture system that allowed the following of the dynamics of the main cell compounds at both hourly and daily time scales. Under N-sufficient conditions, the L:D cycles cause the population to be synchronized, with most of the cells dividing at the beginning of the dark period. The C-specific growth rate was maximal around midday and negative during the dark period due to respiration processes. NL and TC both accumulated during the day and consumed during the night. We showed that NL and TC are highly dynamic compounds, as more than three quarters of NL and TC accumulated during the light period were consumed during the dark period. In contrast to NL, phospholipid and glycolipid to C ratios remained quite stable during the light/dark cycles. The major effect of N starvation on the NL and TC dynamics was to uncouple their diel variations from the L:D cycle, in two different ways depending on their respective role during short-term acclimation. Whereas the TC per cell ratio increased rapidly to reach a stable value in response to N starvation, NL per cell continued to oscillate, but with a pattern out of phase with the L:D cycle.
Microalgal biomass seems to be a promising feedstock for biofuel generation. Microalgae have relative high photosynthetic efficiencies, high growth rates, and some species can thrive in brackish water or seawater and wastewater from the food- and agro-industrial sector. Today, the main interest in research is the cultivation of microalgae for lipids production to generate biodiesel. However, there are several other biological or thermochemical conversion technologies, in which microalgal biomass could be used as substrate. However, the high protein content or the low carbohydrate content of the majority of the microalgal species might be a constraint for their possible use in these technologies. Moreover, in the majority of biomass conversion technologies, carbohydrates are the main substrate for production of biofuels. Nevertheless, microalgae biomass composition could be manipulated by several cultivation techniques, such as nutrient starvation or other stressed environmental conditions, which cause the microalgae to accumulate carbohydrates. This paper attempts to give a general overview of techniques that can be used for increasing the microalgal biomass carbohydrate content. In addition, biomass conversion technologies, related to the conversion of carbohydrates into biofuels are discussed.
Ultraviolet radiation (UVR) is a natural fraction of the solar radiation, and therefore has always influenced life in aquatic
ecosystems. The development of oxygenic photosynthesis 2.5–2.7 billion years ago (Holland 1984) led to drastic chemical changes
in the Earth’s oceans and atmosphere. The gradual increase in photosynthetically produced oxygen over millions of years was
accompanied by a strong enrichment of it in the atmosphere, which ultimately acted as precursor for the ozone (O3) layer in
the stratosphere.
Laboratory cultures are important tools for investigating the biology of microalgae, allowing experimentation under controlled conditions. This control is critical for comparative studies, such as those often used to investigate intra-specific variation in properties of interest. By holding the environment constant, the experimentalist can gain insight into the genetic basis of phytoplankton phenotypes and by extension, into the adaptive history of those genotypes. In most cases the adaptations of interest are those that the algae have evolved in response to their natural environment. However, here it is argued that such experiments may instead reveal evolutionary adaptations to, and/or non-adaptive changes induced by, the culture conditions under which the alga is maintained. We present a review of the processes of evolution as they pertain to microalgal culture, and illustrate this discussion with examples of in-culture evolution from both within and outside the field of phycology. With these considerations in mind, recommendations are made for experimental practice focusing on comparative physiology, for which the effects of in-culture evolution are particularly confounding. Finally we argue that, although problematic in some contexts, the evolutionary propensities of phytoplankton cultures actually present an important opportunity for experimental evolutionary research with direct environmental significance.
We describe a new selection method based on BODIPY (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) staining, fluorescence activated cell sorting (FACS) and microplate-based isolation of lipid-rich microalgae from an environmental sample. Our results show that direct sorting onto solid medium upon FACS can save about 3 weeks during the scale-up process as compared with the growth of the same cultures in liquid medium. This approach enabled us to isolate a biodiverse collection of several axenic and unialgal cultures of different phyla.
The freshwater alga Chlorella, a highly productive source of starch, might substitute for starch-rich terrestrial plants in bioethanol production. The cultivation conditions necessary for maximizing starch content in Chlorella biomass, generated in outdoor scale-up solar photobioreactors, are described. The most important factor that can affect the rate of starch synthesis, and its accumulation, is mean illumination resulting from a combination of biomass concentration and incident light intensity. While 8.5% DW of starch was attained at a mean light intensity of 215 µmol/(m2 s1), 40% of DW was synthesized at a mean light intensity 330 µmol/(m2 s1). Another important factor is the phase of the cell cycle. The content of starch was highest (45% of DW) prior to cell division, but during the course of division, its cellular level rapidly decreased to about 13% of DW in cells grown in light, or to about 4% in those kept in the dark during the division phase. To produce biomass with high starch content, it is necessary to suppress cell division events, but not to disturb synthesis of starch in the chloroplast. The addition of cycloheximide (1 mg/L), a specific inhibitor of cytoplasmic protein synthesis, and the effect of element limitation (nitrogen, sulfur, phosphorus) were tested. The majority of the experiments were carried out in laboratory-scale photobioreactors, where culture treatments increased starch content to up to about 60% of DW in the case of cycloheximide inhibition or sulfur limitation. When the cells were limited by phosphorus or nitrogen supply, the cellular starch content increased to 55% or 38% of DW, respectively, however, after about 20 h, growth of the cultures stopped producing starch, and the content of starch again decreased. Sulfur limited and cycloheximide-treated cells maintained a high content of starch (60% of DW) for up to 2 days. Sulfur limitation, the most appropriate treatment for scaled-up culture of starch-enriched biomass, was carried out in an outdoor pilot-scale experiment. After 120 h of growth in complete mineral medium, during which time the starch content reached around 18% of DW, sulfur limitation increased the starch content to 50% of DW.
Most organic matter can be used for bioenergy generation via anaerobic fermentation. Today, crop plants like maize play the dominant role as substrates for renewable biogas production. In this work we investigated the suitability of six dominant microalgae species (freshwater and saltwater algae and cyanobacteria) as alternative substrates for biogas production. We could demonstrate that the biogas potential is strongly dependent on the species and on the pretreatment. Fermentation of the green alga Chlamydomonas reinhardtii was efficient with a production of 587 ml(±8.8 SE) biogas g volatile solids(-1) (VS(-1)), whereas fermentation of Scenedesmus obliquus was inefficient with only 287 ml(±10.1 SE) biogas g VS(-1) being produced. Drying as a pretreatment decreased the amount of biogas production to ca. 80%. The methane content of biogas from microalgae was 7-13% higher compared to biogas from maize silage. To evaluate integrative biorefinery concepts, hydrogen production in C. reinhardtii prior to anaerobic fermentation of the algae biomass was measured and resulted in an increase of biogas generation to 123% (±3.7 SE). We conclude that selected algae species can be good substrates for biogas production and that anaerobic fermentation can seriously be considered as final step in future microalgae-based biorefinery concepts.
There are currently intensive global research efforts aimed at increasing and modifying the accumulation of lipids, alcohols, hydrocarbons, polysaccharides, and other energy storage compounds in photosynthetic organisms, yeast, and bacteria through genetic engineering. Many improvements have been realized, including increased lipid and carbohydrate production, improved H(2) yields, and the diversion of central metabolic intermediates into fungible biofuels. Photosynthetic microorganisms are attracting considerable interest within these efforts due to their relatively high photosynthetic conversion efficiencies, diverse metabolic capabilities, superior growth rates, and ability to store or secrete energy-rich hydrocarbons. Relative to cyanobacteria, eukaryotic microalgae possess several unique metabolic attributes of relevance to biofuel production, including the accumulation of significant quantities of triacylglycerol; the synthesis of storage starch (amylopectin and amylose), which is similar to that found in higher plants; and the ability to efficiently couple photosynthetic electron transport to H(2) production. Although the application of genetic engineering to improve energy production phenotypes in eukaryotic microalgae is in its infancy, significant advances in the development of genetic manipulation tools have recently been achieved with microalgal model systems and are being used to manipulate central carbon metabolism in these organisms. It is likely that many of these advances can be extended to industrially relevant organisms. This review is focused on potential avenues of genetic engineering that may be undertaken in order to improve microalgae as a biofuel platform for the production of biohydrogen, starch-derived alcohols, diesel fuel surrogates, and/or alkanes.
An analysis of the energy life-cycle for production of biomass using the oil-rich microalgae Nannochloropsis sp. was performed, which included both raceway ponds, tubular and flat-plate photobioreactors for algal cultivation. The net energy ratio (NER) for each process was calculated. The results showed that the use of horizontal tubular photobioreactors (PBRs) is not economically feasible ([NER]<1) and that the estimated NERs for flat-plate PBRs and raceway ponds is >1. The NER for ponds and flat-plate PBRs could be raised to significantly higher values if the lipid content of the biomass were increased to 60% dw/cwd. Although neither system is currently competitive with petroleum, the threshold oil cost at which this would occur was also estimated.
An innovative technology for the biological treatment of wastewater in regions with sufficient solar radiation based on the simultaneous growth and degradation processes of algal and bacterial biomass is presented. The aim of the work is the improvement of pond technology through the formation of stable algae-bacteria aggregates, which a) permit a simple separation of the algal biomass by gravity sedimentation, b) enable a high removal efficiency for organic carbon and nutrients, and c) are independent in terms of oxygen provision through algal photosynthesis. Algae-bacteria aggregates could be developed with a suitable algal species (Chlorella vulgaris, Strain Hamburg) as a 'model organism' in a wastewater environment. The morphology of algal-bacterial flocs is similar to activated sludge flocs. They are stable and settle quickly. Floc size ranged between 400 and 800 microm. Results of our experiments with an artificially irradiated lab-scale system, operated in continuous flow mode, revealed that even at a relatively short hydraulic detention time of two days, a high elimination capacity of 9.96 g N m(-2) d(-1) and 0.87g Pm(-2) d(-1) can be achieved. Recent investigations confirmed that floc formation of unicellular algae and wastewater bacteria also could be developed and maintained in a pilot-scale system with a water depth of 0.5 m.
Large-scale production of microalgal storage compounds will likely involve marine microalgae. Previously, we described a method to enrich microalgae with a high storage compound productivity from a natural inoculum. Here, this strategy was implemented under marine conditions in a sequencing batch reactor. The influence of the volume exchange ratio and the moment of ammonium addition in the day-night cycle on the storage compound productivity are described. Storage compound productivity was maximal if ammonium was supplied at the start of the dark period rather than the light period, irrespective of the volume exchange ratio. Increasing the volume exchange ratio from 33 to 50 % per cycle induced a decrease in storage compound production if ammonium was supplied in the light whereas the storage compound productivity was comparable when ammonium was supplied in the dark. The latter indicates a shift of cell division processes to the light period at increasing volume exchange ratio, although ammonium uptake completely occurred in the dark period.
Several challenges have to be overcome for the production of biofuel from algae to be commercialized. Key among those challenges is the management of contamination in open ponds; without technology to do this, biomass cannot be produced economically. A number of successful open pond technologies have been developed that manage contamination by creating an extreme environment that limits the amount of contamination and improves the stability of these systems. However, these solutions are neither aligned with the economics of fuel production, which demand low costs and high volumes, nor are they suited to many strains of algae considered candidates for fuel production. This work describes a possible solution developed by Sapphire Energy, Inc., to the contamination challenge of open pond cultivation with a strain of algae considered suitable for the production of biofuel. This example illustrates an integrated solution that incorporates pest identification, tracking, and management, and it focuses on a primitive fungal pest that has been shown to crash open ponds of algae used for the production of biofuels. The implementation of this solution can prevent these crashes and offers an approach to managing contamination in open ponds, thereby advancing the prospect of using algae as an economically viable strategy for the production of biofuels.
To improve the economic viability of microalgal biodiesel, it will be essential to optimize the productivity of fuel molecules
such as triacylglyceride (TAG) within the microalgal cell. To understand some of the triggers required for the metabolic switch
to TAG production, we studied the effect of the carbon supply (acetate or CO2) in Chlamydomonas reinhardtii (wild type and the starchless sta6 mutant) grown under low N availability. As expected, initial rates of TAG production were much higher when acetate was present
than under strictly photosynthetic conditions, particularly for the sta6 mutant, which cannot allocate resources to starch. However, in both strains, TAG production plateaued after a few days in
mixotrophic cultures, whereas under autotrophic conditions, TAG levels continued to rise. Moreover, the reduced growth of
the sta6 mutant meant that the greatest productivity (measured as mg TAG liter−1 day−1) was found in the wild type growing autotrophically. Wild-type cells responded to low N by autophagy, as shown by degradation
of polar (membrane) lipids and loss of photosynthetic pigments, and this was less in cells supplied with acetate. In contrast,
little or no autophagy was observed in sta6 mutant cells, regardless of the carbon supply. Instead, very high levels of free fatty acids were observed in the sta6 mutant, suggesting considerable alteration in metabolism. These measurements show the importance of carbon supply and strain
selection for lipid productivity. Our findings will be of use for industrial cultivation, where it will be preferable to use
fast-growing wild-type strains supplied with gaseous CO2 under autotrophic conditions rather than require an exogenous supply of organic carbon.
Successful algal biomass cultivation at scale is a key limiting step for the production of algal biofuels and other high-value products, and crop protection against undesirable biomass losses will be a critically important component of commercialization efforts. If algal biomass production occurs in large, open, outdoor ponds, then it can be expected that invasions of these production facilities by multiple species of algae, invertebrate herbivores, and pathogens will only be a matter of time. This review identifies and discusses key aspects of community and ecosystem ecology that have direct relevance to the successful cultivation of algal biomass. We use experiences and examples from commercial agriculture to illustrate core ecological principles of crop cultivation that we believe can successfully be transferred to large-scale algal biomass production. We then discuss the degree to which herbivores and disease can significantly reduce potential yields, and the concepts of biological control. We also discuss the effects of crop species diversity and composition on algal biomass production, and explore the potential benefits of algal polycultures in large-scale algal biomass cultivation systems.
Algal biofuel production offers great potential as a sustainable source of bioenergy without competing for arable land with food crops. However, many challenges must be overcome to enable this to be done commercially at the scale required to produce biofuels. Here we explain how an understanding of algal ecology could lead to more reliable raceway-based microalgal cultivation, drawing from established principles of community ecology to highlight practices that could be applied to protect algal cultures from unwanted contaminants. Using theoretical concepts, we show how an understanding of local community dynamics at the species level might be used to enhance productivity by encouraging certain community structures over others.
Mixed culture biotechnology (MCB) could become an attractive addition or alternative to traditional pure culture based biotechnology for the production of chemicals and/or bioenergy. On the basis of ecological selection principles, MCB-based processes can be established that generate a narrow product spectrum from a mixed substrate. Three example processes are briefly discussed in this paper: anaerobic digestion aimed at the production of methane-containing biogas, mixed culture fermentation for the production of solvents or biohydrogen, and a two-step process for the production of polyhydroxyalkanoates. These examples give an idea of the potential contribution of mixed culture biotechnology to sustainable production of bioenergy from waste.
It is well-established that microalgal-derived biofuels have the potential to make a significant contribution to the US fuel market, due to several unique characteristics inherent to algae. Namely, autotrophic microalgae are capable of achieving very high efficiencies in converting solar energy into biomass and oil relative to terrestrial oilseed crops, while at the same time exhibiting great flexibility in the quality of land and water required for algal cultivation. These characteristics allow for the possibility to produce appreciable amounts of algal biofuels relative to today’s petroleum fuel market, while greatly mitigating “food-versus-fuel” concerns. However, there is a wide lack of public agreement on the near-term economic viability of algal biofuels, due to uncertainties and speculation on process scale-up associated with the nascent stage of the algal biofuel industry.
Microalgae-derived lipids in the form of triacylglycerols (TAGs) are considered an alternative resource for the production of biofuels and food commodities. Large scale production of microalgal TAGs is currently uneconomical. The cost price could be reduced by improving the areal and volumetric TAG productivity. The economic value could be increased by enhancing the TAG quality. To improve these characteristics, the impact of light intensity, and the combined impact of pH and temperature on TAG accumulation were studied for Scenedesmus obliquus UTEX 393 under nitrogen starved conditions. The maximum TAG content was independent of light intensity, but varied between 18% and 40% of dry weight for different combinations of pH and temperature. The highest yield of fatty acids on light (0.263g/mol photon) was achieved at the lowest light intensity, pH 7 and 27.5°C.
Among the technologies being examined to produce renewable fuels, microalgae are viewed by many in the scientific community as having the greatest potential to become economically viable. Algae are capable of producing greater than 50,000kg/acre/year of biomass [1]. Additionally, most algae naturally accumulate energy-dense oils that can easily be converted into transportation fuels. To reach economic parity with fossil fuels there are still several challenges. These include identifying crop protection strategies, improving harvesting and oil extraction processes, and increasing biomass productivity and oil content. All of these challenges can be impacted by genetic, molecular, and ultimately synthetic biology techniques, and all of these technologies are being deployed to enable algal biofuels to become economically competitive with fossil fuels.
Microalgae are among the most diverse organisms on the planet, and as a result of symbioses and evolutionary selection, the configuration of core metabolic networks is highly varied across distinct algal classes. The differences in photosynthesis, carbon fixation and processing, carbon storage, and the compartmentation of cellular and metabolic processes are substantial and likely to transcend into the efficiency of various steps involved in biofuel molecule production. By highlighting these differences, we hope to provide a framework for comparative analyses to determine the efficiency of the different arrangements or processes. This sets the stage for optimization on the based on information derived from evolutionary selection to diverse algal classes and to synthetic systems.
Polyhydroxyalkanoates (PHAs) are microbial storage polymers that attract interest as bioplastics. PHAs can be produced with open mixed cultures if a suitable enrichment step based on the ecological role of PHA is used. An acetate-fed sequencing batch reactor operated with 1 day biomass residence time and with feast-famine cycles of 12 h was used to enrich a mixed culture of PHA producers. In subsequent fed-batch experiments under growth limiting conditions, the enriched mixed culture produced PHA up to a cellular content of 89 wt % within 7.6 h (average rate of 1.2 g/g/h). The PHA produced from acetate was the homopolymer polyhydroxybutyrate. The culture was dominated by a Gammaproteobacterium that showed little similarity on 16S rRNA level with known bacteria (<90% sequence similarity). The mixed culture process for PHA production does not require aseptic conditions. Waste streams rather than pure substrates could be used as raw materials.
Modern life is intimately linked to the availability of fossil fuels, which continue to meet the world's growing energy needs even though their use drives climate change, exhausts finite reserves and contributes to global political strife. Biofuels made from renewable resources could be a more sustainable alternative, particularly if sourced from organisms, such as algae, that can be farmed without using valuable arable land. Strain development and process engineering are needed to make algal biofuels practical and economically viable.
Microalgae are a promising alternative source of lipid for biodiesel production. One of the most important decisions is the
choice of species to use. High lipid productivity is a key desirable characteristic of a species for biodiesel production.
This paper reviews information available in the literature on microalgal growth rates, lipid content and lipid productivities
for 55 species of microalgae, including 17 Chlorophyta, 11 Bacillariophyta and five Cyanobacteria as well as other taxa. The
data available in the literature are far from complete and rigorous comparison across experiments carried out under different
conditions is not possible. However, the collated information provides a framework for decision-making and a starting point
for further investigation of species selection. Shortcomings in the current dataset are highlighted. The importance of lipid
productivity as a selection parameter over lipid content and growth rate individually is demonstrated.
The interaction of whole cell metabolism with the distribution of excitation energy between photosystem 2 (PS2) and photosystem 1 (PS1), the light state transition, was investigated in vivo in the green alga Selenastrumminutum. Nitrogen limited cells of S. minutum were presented with a pulse of either NH4+ or NO3− in the light. As shown previously, CO2 fixation is inhibited and high rates of N assimilation ensue [(1986) Plant Physiol. 81, 273–279]. NH4+ assimilation has a much higher requirement ratio for ATP/NADPH than either CO2 or NO3− assimilation and thus drastically increases the demand for ATP relative to reducing power. Room temperature chlorophyll a fluorescence kinetic measurements showed that a reversible non-photochemical quenching of PS2 fluorescence accompanied the assimilation of NH4+ but not the assimilation of NO3− or CO2. 77K fluorescence emission spectra taken from samples removed at regular intervals during NH+4assimilation showed that the non-photochemical quenching of PS2 was accompanied by a complementary increase in the fluorescence yield of PS1, characteristic of a transition to state 2. Our data suggests that S. minutum responds to the increased demand for ATP/NADPH during NH4 assimilation by inducing the light state transition to direct more excitation energy to PS1 at the expense of PS2 to increase the production of ATP by cyclic electron transport.
In her News Feature “Biotech’s green gold”, Emily Waltz details the ‘hype’ being propagated around emerging microalgal biofuel technologies, which often exceeds the physical and thermodynamic constraints that ultimately define their economic viability. Our calculations counter such excessive claims and demonstrate that 22 MJ m−2 d−1 solar radiation supports practical yield maxima of ∼60 to 100 kl oil ha−1 y−1 (∼6,600 to 10,800 gal ac−1 y−1) and an absolute theoretical ceiling of ∼94 to 155 kl oil ha−1 y−1, assuming a maximum photosynthetic conversion efficiency of 10%. To evaluate claims and provide an accurate analysis of the potential of microalgal biofuel systems, we have conducted industrial feasibility studies and sensitivity analyses based on peer-reviewed data and industrial expertise. Given that microalgal biofuel research is still young and its development still in flux, we anticipate that the stringent assessment of the technology's economic potential presented below will assist R&D investment and policy development in the area going forward.
Polyhydroxyalkanoates (PHAs) are microbial storage polymers that attract interest as bioplastics. PHAs can be produced with open mixed cultures if a suitable enrichment step based on the ecological role of PHA is used. An acetate-fed sequencing batch reactor operated with 1 day biomass residence time and with feast-famine cycles of 12 h was used to enrich a mixed culture of PHA producers. In subsequent fed-batch experiments under growth limiting conditions, the enriched mixed culture produced PHA up to a cellular content of 89 wt % within 7.6 h (average rate of 1.2 g/g/h). The PHA produced from acetate was the homopolymer polyhydroxybutyrate. The culture was dominated by a Gammaproteobacterium that showed little similarity on 16S rRNA level with known bacteria (<90% sequence similarity). The mixed culture process for PHA production does not require aseptic conditions. Waste streams rather than pure substrates could be used as raw materials.
Production of polyhydroxyalkanoates (PHA) by mixed cultures has been widely studied in the last decade. Storage of PHA by mixed microbial cultures occurs under transient conditions of carbon or oxygen availability, known respectively as aerobic dynamic feeding and anaerobic/aerobic process. In these processes, PHA-accumulating organisms, which are quite diverse in terms of phenotype, are selected by the dynamic operating conditions imposed to the reactor. The stability of these processes during long-time operation and the similarity of the polymer physical/chemical properties to the one produced by pure cultures were demonstrated. This process could be implemented at industrial scale, providing that some technological aspects are solved. This review summarizes the relevant research carried out with mixed cultures for PHA production, with main focus on the use of wastes or industrial surplus as feedstocks. Basic concepts, regarding the metabolism and microbiology, and technological approaches, with emphasis on the kind of feedstock and reactor operating conditions for culture selection and PHA accumulation, are described. Challenges for the process optimization are also discussed.
Thirty microalgal strains were screened in the laboratory for their biomass productivity and lipid content. Four strains (two marine and two freshwater), selected because robust, highly productive and with a relatively high lipid content, were cultivated under nitrogen deprivation in 0.6-L bubbled tubes. Only the two marine microalgae accumulated lipid under such conditions. One of them, the eustigmatophyte Nannochloropsis sp. F&M-M24, which attained 60% lipid content after nitrogen starvation, was grown in a 20-L Flat Alveolar Panel photobioreactor to study the influence of irradiance and nutrient (nitrogen or phosphorus) deprivation on fatty acid accumulation. Fatty acid content increased with high irradiances (up to 32.5% of dry biomass) and following both nitrogen and phosphorus deprivation (up to about 50%). To evaluate its lipid production potential under natural sunlight, the strain was grown outdoors in 110-L Green Wall Panel photobioreactors under nutrient sufficient and deficient conditions. Lipid productivity increased from 117 mg/L/day in nutrient sufficient media (with an average biomass productivity of 0.36 g/L/day and 32% lipid content) to 204 mg/L/day (with an average biomass productivity of 0.30 g/L/day and more than 60% final lipid content) in nitrogen deprived media. In a two-phase cultivation process (a nutrient sufficient phase to produce the inoculum followed by a nitrogen deprived phase to boost lipid synthesis) the oil production potential could be projected to be more than 90 kg per hectare per day. This is the first report of an increase of both lipid content and areal lipid productivity attained through nutrient deprivation in an outdoor algal culture. The experiments showed that this marine eustigmatophyte has the potential for an annual production of 20 tons of lipid per hectare in the Mediterranean climate and of more than 30 tons of lipid per hectare in sunny tropical areas.
Two laboratory-scale sequencing batch reactors (SBRs) were operated for enhanced biological phosphorus removal (EBPR) in alternating anaerobic-aerobic or alternating anaerobic-anoxic modes, respectively. Polyphosphate-accumulating organisms (PAOs) were enriched in the anaerobic-aerobic SBR and denitrifying PAOs (DPAOs) were enriched in the anaerobic-aerobic SBR. Fluorescence in situ hybridization (FISH) demonstrated that the well-known PAO, "Candidatus Accumulibacter phosphatis" was abundant in both SBRs, and post-FISH chemical staining with 4,6-diamidino-2-phenylindol (DAPI) confirmed that they accumulated polyphosphate. When the anaerobic-anoxic SBR enriched for DPAOs was converted to anaerobic-aerobic operation, aerobic uptake of phosphorus by the resident microbial community occurred immediately. However, when the anaerobic-aerobic SBR enriched for PAOs was exposed to one cycle with anoxic rather than aerobic conditions, a 5-h lag period elapsed before phosphorus uptake proceeded. This anoxic phosphorus-uptake lag phase was not observed in the subsequent anaerobic-aerobic cycle. These results demonstrate that the PAOs that dominated the anaerobic-aerobic SBR biomass were the same organisms as the DPAOs enriched under anaerobic-anoxic conditions.
Microalgae represent an exceptionally diverse but highly specialized group of micro-organisms adapted to various ecological habitats. Many microalgae have the ability to produce substantial amounts (e.g. 20-50% dry cell weight) of triacylglycerols (TAG) as a storage lipid under photo-oxidative stress or other adverse environmental conditions. Fatty acids, the building blocks for TAGs and all other cellular lipids, are synthesized in the chloroplast using a single set of enzymes, of which acetyl CoA carboxylase (ACCase) is key in regulating fatty acid synthesis rates. However, the expression of genes involved in fatty acid synthesis is poorly understood in microalgae. Synthesis and sequestration of TAG into cytosolic lipid bodies appear to be a protective mechanism by which algal cells cope with stress conditions, but little is known about regulation of TAG formation at the molecular and cellular level. While the concept of using microalgae as an alternative and renewable source of lipid-rich biomass feedstock for biofuels has been explored over the past few decades, a scalable, commercially viable system has yet to emerge. Today, the production of algal oil is primarily confined to high-value specialty oils with nutritional value, rather than commodity oils for biofuel. This review provides a brief summary of the current knowledge on oleaginous algae and their fatty acid and TAG biosynthesis, algal model systems and genomic approaches to a better understanding of TAG production, and a historical perspective and path forward for microalgae-based biofuel research and commercialization.
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