Biomass and Biofuels from Microalgae

Microalgae cultivation is a promising methodology for solving some of the future problems of biomass production (i.e. renewable food, feed and bioenergy production). There is no doubt that in conjunction with conventional growth systems, novel technologies must be developed in order to produce the large-scale sustainable microalgae products. Here, we review some of the most promising existing microalgae biomass growth technologies and summarise some of the novel methodologies for sustainable microalgae production.

Immobilized cells entrapped within a polymer matrix or attached onto the surface of a solid support have advantages over their free-cell counterpart, with easier harvesting of the biomass, enhanced wastewater treatment, and enriched bioproduct generation. Immobilized microalgae have been used for a diverse number of bioprocesses including gaining access to high-value products (biohydrogen, biodiesel, and photopigments), removal of nutrients (nitrate, phosphate, and ammonium ions), heavy metal ion removal, biosensors, and stock culture management. Wastewater treatment processes appear to be one of the most promising applications for immobilized microalgae, which mostly involve heavy metal and nutrient removal from liquid effluents. This chapter outlines the current applications of immobilized microalgae with an emphasis on alternative immobilization approaches. Advances in immobilization processes and possible research directions are also highlighted.

The current prices of microalgae oils are much higher than oils from higher plants (vegetable oils) mainly due to the high cost of photoautotrophic cultivation of microalgae. However, many strains of microalgae can also grow and produce oil using organic carbons, as the carbon source under dark (heterotrophy) or light conditions (mixotrophy). Lipid productivities of most strains of microalgae are higher in culture systems that incorporate heterotrophic metabolisms (presence of organic carbon source) than under photoautotrophic conditions. This is because for many strains, cell growth rates and final cell concentrations are higher in heterotrophic cultures than in photoautotrophic cultures. Furthermore, in some cases, the oil contents of the cells are also higher in cultures incorporating heterotrophic metabolisms. It has also been reported for some strains that the quality of oil produced in the presence of organic carbon sources are more suitable for biodiesel oil production than those produced under photoautotrophic conditions. Thus, heterotrophy can be used to reduce the cost of biodiesel oil production, but the effectiveness of the various organic carbons in supporting cell growth and oil accumulation depends on the strain and other culture conditions. Use of wastewaters for cultivation of microalgae can further substantially reduce the cost of production (since they contain carbon, nitrogen, and other nutrients) and also reduce the requirement for freshwater. Generally, many factors such as nitrogen limitation, phosphate limitation, silicon limitation, control of pH, and low temperature can be used to increase oil accumulation, although their effectiveness depends on the strain and other culture conditions.

The feasibility of using various culture systems incorporating heterotrophic metabolism for biodiesel oil production was compared. Heterotrophic culture can be used to achieve high cell concentration, and depending on the strain and organic carbon source employed, the introduction of light (mixotrophic culture) can enhance cell growth and oil accumulation. However, mixotrophic cultures also face the problem of light limitation, and depending on the relative concentrations of the organic carbon source and light intensity, the interaction between the heterotrophic and photoautotrophic metabolic activities can have negative effects on cell growth and oil accumulation. Systems that separate the two metabolic activities in time or space, such as cyclic photoautotrophic–heterotrophic cultures, sequential heterotrophic–photoautotrophic cultures, and sequential photoautotrophic–mixotrophic cultures, can all be used to improve oil productivity. However, the effectiveness of each system depends on the strain of microalgae and other culture conditions.

Production of microalgal biomass requires large amounts of nitrogen (N) and phosphorus (P). The sustainability and economic viability of microalgae production could be significantly improved if N and P are not supplied by synthetic fertilizers but with wastewater. Microalgae already play an important role in wastewater treatment, yet several challenges remain to optimally convert wastewater nutrients into microalgal biomass. This book chapter aims to give an overview of the potential of using wastewater for microalgae production, as well some challenges that should be taken into account. We also review the benefits of combining microalgal biomass production with wastewater treatment.

This chapter develops the principles and rationale for an algae-based biofuel production coupled to bioremediation of municipal and agricultural wastewaters. A synergistic model for algal wastewater treatment is proposed, which addresses several economic bottlenecks to earlier algal systems and promotes value-added products, including a high-quality effluent in addition to biodiesel to improve the economic feasibility of algal biofuels. Finally, we review candidate species for full-scale algae production ponds based on algal structure, physiology and ecology, and methods for extraction of algal oils for biodiesel production and coproducts. The dominant strains of algae that are commonly found in wastewater ponds, including Euglenia, Scenedesmus, Selenastrum, Chlorella, and Actinastrum, are suggested as candidates for large-scale culturing based on their ability to strip nutrients and organic matter from wastewater, grow rapidly, and produce a significant level of algal oil. Oil extraction by supercritical fluid extraction (SFE) is discussed as an efficient means of isolating algal oil and other commercially important high-value compounds from algal biomass. Together with water and CO₂ reclamation, such products may shift the economics of algal biomass production to allow production of low-value commodities including biodiesel and biogas.

Various microalgae species have shown a differential ability to bioremediate atmospheric CO₂. This chapter reports biomass concentration, biomass productivity, and CO₂ fixation rates of several microalgae and cyanobacteria species under different CO₂ concentrations and culture conditions. Research indicates that microalgal species of Scenedesmuss obliquss, Duniella tertiolecta, Chlorella vulgaris, Phormidium sp., Amicroscopica negeli, and Chlorococcum littorale are able to bioremediate CO₂ more effectively than other species. Furthermore, coccolithophorid microalgae such as Chrysotila carterae were also found to effectively bioremediate CO₂ into organic biomass and generate inorganic CaCO₃ as additional means of removing atmospheric CO₂. Important factors to increase the rate of CO₂ bioremediation such as initial cell concentration, input CO₂ concentration, and aeration rate are reviewed and discussed.

Synthetic Biology is an interdisciplinary approach combining biotechnology, evolutionary biology, molecular biology, systems biology and biophysics. While the exact definition of Synthetic Biology might still be debatable, its focus on design and construction of biological devices that perform useful functions is clear and of great utility to engineering algae. This relies on the re-engineering of biological circuits and optimization of certain metabolic pathways to reprogram algae and introduce new functions in them via the use of genetic modules. Genetic editing tools are primary enabling techniques in Synthetic Biology and this chapter discusses common techniques that show promise for algal gene editing. The genetic editing tools discussed in this chapter include RNA interference (RNAi) and artificial microRNAs, RNA scaffolds, transcription activator-like effector nucleases (TALENs), RNA guided Cas9 endonucleases (CRISPR), and multiplex automated genome engineering (MAGE). DNA and whole genome synthesis is another enabling technology in Synthetic Biology and might present an alternative approach to drastically and readily modify algae. Clear and powerful examples of the potential of whole genome synthesis for algal engineering are presented. Also, the development of relevant computational tools, and genetic part registries has stimulated further advancements in the field and their utility in algal research and engineering is described. For now, the majority of synthetic biology efforts are focused on microbes as many pressing problems, such as sustainability in food and energy production rely on modification of microorganisms. Synthetic modifications of algal strains to enhance desired physiological properties could lead to improvements in their utility.

The uniquely diverse metabolism of algae can make this group of organisms a prime target for biotechnological purposes and applications. To fully reap their biotechnological potential, molecular genetic techniques for manipulating algae must gain track and become more reliable. To this end, this chapter describes the currently available molecular genetic techniques and resources, as well as a number of relevant computational tools that can facilitate genetic manipulation of algae. Genetic transformation is perhaps the most elemental of such techniques and has become a well-established approach in algal-based genetic experiments. The utility of genetic transformations and other molecular genetic techniques is guided by phenotypic insights resulting from forward and reverse genetic analysis. As such, genetic transformations can form the building blocks for more complex genic manipulations. Herein, we describe currently available engineered homologous recombination or recombineering approaches, which allow for substitutions, insertions, and deletions of larger DNA segments, as well as manipulation of endogenous DNA. In addition, as reagent resources in the form of cloned open reading frames (ORFs) of transcription factors (TFs) and metabolic enzymes become more readily available, algal genetic manipulations can greatly increase the range of obtainable phenotypes for biotechnological applications. Such resources and a few case studies are highlighted in the context of candidate genes for algal bioengineering. On a final note, tools for computer-aided design (CAD) to prototype molecular genetic techniques and protocols are described. Such tools could greatly increase the reliability and efficiency of genetic molecular techniques for algal bioengineering.

Genomic sequencing is the first step in a systems level study of an algal species, and sequencing studies have grown steadily in recent years. Completed sequences can be tied to algal phenotypes at a systems level through constructing genome-scale metabolic network models. Those models allow the prediction of algal phenotypes and genetic or metabolic modifications, and are constructed by tying the genes to reactions using enzyme databases, then representing those reactions in a concise mathematical form by means of stoichiometric matrices. This is followed by experimental validation using gene deletion or proteomics and metabolomics studies that may result in adding reactions to the model and filling phenotypic gaps. In this chapter, we offer a summary of completed and ongoing algal genomic projects before proceeding to holistically describing the process of constructing genome-scale metabolic models. Relevant examples of algal metabolic models are presented and discussed. The analysis of an alga’s emergent properties from metabolic models is also demonstrated using flux balance analysis (FBA) and related constraint-based approaches to optimize a given metabolic phenotype, or sets of phenotypes such as algal biomass. We also summarize readily available optimization tools rooted in constraint-based modeling that allow for optimizing bioproduction and algal strains. Examples include tools used to develop knockout strategies, identify optimal bioproduction strains, analyze gene deletions, and explore functional relationships within sets in a metabolic model. All in all, this systems level approach can lead to a better understanding and prediction of algal metabolism leading to more robust and cheaper applications.

An advanced understanding of the genetics of microalgae and the availability of molecular biology tools are both critical to the development of advanced strains, which offer efficiency advantages for primary production, and more specifically in the context of production for biocrude and renewable energy. Consequently, we outline the current state of the art in microalgal molecular biology including the available genome sequences, molecular techniques and toolkits, amenable strains for transformation of nuclear and plastid genomes, and the control of transgenes at both transcriptional and translational levels. We also examine some strategies for improvement of expression and regulation. We suggest the primary strategies in strain improvement that are most relevant to biocrude applications; briefly illustrate the process of photosynthesis to enable identification of targets for improvement of net photosynthetic conversion efficiency in mass cultivation; and further discuss how improvement of metabolic systems may also be achieved and benefit production models. Finally, we acknowledge the aspects of prudent risk assessment and consequent regulation that are developing and how our knowledge of natural algae in existing ecosystems, and GM work in conventional agriculture both contribute lessons to these discussions. We conclude that if properly managed, these developments provide significant potential for increasing global capacity for renewable fuel production from microalgae and that these developments could also have benefits for other applications.

Large-scale production of microalgae for biofuels is still facing several major challenges to become competitive with other forms of renewable and non-renewable energy. A major challenge is harvesting, which requires the separation of a low amount of biomass consisting of small individual cells from a large volume of culture medium. Flocculation is seen as a promising low-cost harvesting method for microalgae biomass. In this chapter, the challenges and potential advantages of using flocculation as a harvesting method for microalgae are reviewed.

Industrial-scale microalgae production will likely require large energy-intensive technologies for both culture and biomass recovery; energy-efficient and cost-effective microalgae dewatering and water management are major challenges. Primary dewatering is typically achieved through flocculation followed by separation via settling or flotation. Flocculants are relatively expensive, and their presence can limit the reuse of de-oiled flocculated microalgae. Natural flocculation of microalgae—autoflocculation—occurs in response to changes in pH and water hardness and, if controlled, might lead to less-expensive “flocculant-free” dewatering. A better understanding of autoflocculation should also prompt higher yields by preventing unwanted autoflocculation. Autoflocculation is driven by double-layer coordination between microalgae, Ca⁺² and Mg⁺², and/or mineral surface precipitates of calcite, Mg(OH)₂, and hydroxyapatite that form primarily at pH > 8. Combining surface complexation models that describe the interface of microalgae:water, calcite:water, Mg(OH)₂:water, and hydroxyapatite:water allows optimal autoflocculation conditions—for example pH, Mg, Ca, and P levels—to be identified for a given culture medium.

Harvesting of dilute cultures of algae from large volumes of culture needed for production of biofuels and bioproducts is a substantial hurdle to the economic viability of algal biofuels. While centrifugation and sedimentation are already scaled to volumes that would allow direct application to algal biofuel production, their economics to the production of biofuel are not favorable. The industry has reevaluated the existing technologies and continues to innovate around the harvesting of microalgae for biofuels and bioproducts. This review discusses the historical approaches and recent advances while comparing and contrasting the different methods. An engineering estimate of comparative costs is also provided.

Chemical energy can be produced from solar energy via photosynthesis. Solar energy can also be converted into electricity via photovoltaic devices. These two mechanisms would seem to compete for the same resources. However, due to differences in the spectral requirements, there is an opportunity to coproduce both electricity and chemical energy from a single facility. We propose to introduce an active filter or solar panel above a microalgae pond to generate both electricity and chemical energy. There are several advantages to such technology including reduced heating (saving freshwater) and an independent electricity supply. Additionally, by channeling targeted illumination back into the microalgae ponds, we can double the amount of light absorbed by the microalgae. This can result in increased biomass productivity.

Anaerobic digestion offers a potential pathway to eliminate some of the overheads for microalgae-based biofuels bio-refinery production systems. It is anticipated that the incorporation and integration of anaerobic digestion with microalgae-based biofuels production is able to attain higher efficiency and improve sustainability in the production of biofuels from microalgae. This chapter investigates several of the technical issues associated with anaerobic digestion of microalgae biomass including the low concentration of biodegradable (digestible) microalgae substrates, cell wall disruption and high lipid concentrations. Also highlighted is when the incorporation of anaerobic digestion into a biofuels bio-refinery concept, several anaerobic digestion-related issues can be addressed by the pre-treatment methods used to process microalgae for liquid and gaseous biofuels. This chapter also discusses other technical issues associated with the anaerobic digestion of microalgae including ammonia inhibition, low C/N ratio and co-digestion. Gas produced by the anaerobic digestion of residual microalgae biomass can be used for electrical or thermal energy within the microalgae biofuels bio-refinery, while the high density microalgae cultures can provide efficient biogas purification. The resulting digestate has been shown to be an ideal nutrient source for the continued growth of additional microalgae biomass, and helps to close the nutrient loop associated with large-scale microalgae biomass production. With a greater understanding of the different microalgae species and their characteristics, the anaerobic digestion of microalgae and their residues must be optimised to play an essential role in the sustainable future of clean energy derived from microalgae biomass.

Microalgae biofuels have been under development for the last 40 years; however, in the last 6 years, this development has intensified due to higher oil prices and wider acceptance of anthropogenic climate change. Despite the excellent potential of algal biofuels, they are not yet commercially viable. The reason for this lack of progress is examined in this chapter by firstly reviewing the range of different technology options for biofuels from microalgae. Secondly, an analysis of the available techno-economic and energy assessments is performed highlighting the effect that each system element has on the overall viability.