Association with an ammonium-excreting bacterium allows diazotrophic culture of oil-rich eukaryotic microalgae

Concerns regarding the depletion of the world's reserves of oil and global climate change have promoted an intensification of research and development toward the production of biofuels and other alternative sources of energy during the last years. There is currently much interest in developing the technology for third-generation biofuels from microalgal biomass mainly because of its potential for high yields and reduced land use changes in comparison with biofuels derived from plant feedstocks. Regardless of the nature of the feedstock, the use of fertilizers, especially nitrogen, entails a potential economic and environmental drawback for the sustainability of biofuel production. In this work, we have studied the possibility of nitrogen biofertilization by diazotrophic bacteria applied to cultured microalgae as a promising feedstock for next-generation biofuels. We have obtained an Azotobacter vinelandii mutant strain that accumulates several times more ammonium in culture medium than wild-type cells. The ammonium excreted by the mutant cells is bioavailable to promote the growth of nondiazotrophic microalgae. Moreover, this synthetic symbiosis was able to produce an oil-rich microalgal biomass using both carbon and nitrogen from the air. This work provides a proof of concept that artificial symbiosis may be considered an alternative strategy for the low-N-intensive cultivation of microalgae for the sustainable production of next-generation biofuels and other bioproducts.     

Potential biotechnological application of microalgae: a critical review

Microalgae are diverse microorganisms inhabiting a wide range of habitats with only a small fraction being cultivated for human use. Recently, interest in microalgal research has increased in the quest for alternative renewable fuels due to possible depletion of fossil fuels in the near future. However, costly downstream processing has hampered the commercialization of biofuels derived from microalgae. Several value added products of industrial, pharmaceutical and agricultural relevance could be simultaneously derived from microalgae during bioenergy production. Despite these value-added products having the potential to offset the high cost of downstream processing of renewable fuels, their production has not been explored in-depth. This review presents a critical overview of the current state of biotechnological applications of microalgae for human benefit and highlights possible areas for further research and development.     

Selection, breeding and engineering of microalgae for bioenergy and biofuel production

Microalgal production technologies are seen as increasingly attractive for bioenergy production to improve fuel security and reduce CO(2) emissions. Photosynthetically derived fuels are a renewable, potentially carbon-neutral and scalable alternative reserve. Microalgae have particular promise because they can be produced on non-arable land and utilize saline and wastewater streams. Furthermore, emerging microalgal technologies can be used to produce a range of products such as biofuels, protein-rich animal feeds, chemical feedstocks (e.g. bioplastic precursors) and higher-value products. This review focuses on the selection, breeding and engineering of microalgae for improved biomass and biofuel conversion efficiencies.     

Current Large-Scale US Biofuel Potential from Microalgae Cultivated in Photobioreactors

Current assessments of the commercial viability and productivity potential of microalgae biofuels have been forced to extrapolate small-scale research data. The resulting analyses are not representative of microalgae cultivation and processing at industrial scale. To more accurately assess the current near-term realizable, large-scale microalgae productivity potential in the USA, this paper presents a model of microalgae growth derived from industrial-scale outdoor photobioreactor growth data. This model is combined with thermal models of the photobioreactor system and 15?years of hourly historical weather data from 864 locations in the USA to more accurately assess the current productivity potential of microalgae. The resulting lipid productivity potential of Nannochloropsis is presented in the form of a map that incorporates various land availability models to illustrate the near-term feasible cultivation locations and corresponding productivity potentials for the USA. The discussion focuses on a comparison of model results with productivity potentials currently reported in literature, an assessment demonstrating the scale of Department of Energy 2030 alternative fuel goals, and a critical comparison of productivity potential in several key regions of the USA.     

Conversion of CO2 into biomass by microalgae: how realistic a contribution may it be to significant CO2 removal?

Microalgae have been proposed as a CO₂ removal option to contribute to climate change avoidance and problems coming from the use of fossil fuels. However, even though microalgae can be used to fix CO₂ from air or flue gases, they do not permit long-term CO₂ storage because they are easily decomposed. On the other hand, microalgae can contribute to an enhancement in human sustainability by producing biofuels as an alternative to fossil fuels in addition to the production of other useful chemicals and commodities. Moreover, microalgae can contribute to enhancing the sustainability of waste treatment processes, reducing the energy consumed, and improving the recycling of nutrients contained within them. This paper reviews the potential contribution of these processes and the existing knowledge in these areas.     

Microalgae potential as a biogas source: current status,restraints and future trends

In recent years, the world energy demands have had a recurrent increase. For this reason the alternative to the fossil fuel resources are trend topics in investigation. Microalgae have been extensively studied as a source of biofuels and as one of the most promising alternatives in this new framework. One of the possibilities of obtaining renewable energy from microalgae is biogas production using anaerobic digestion process. This process is considered a significant component for biofuels and waste management, since represent an opportunity for energy generation using different wastewater products; also, the economic viability of microalgae liquid biofuel production could be improved. However, the anaerobic digestion of microalgae biomass is still not optimized because of the numerous technical limitations such as the microalgae characteristics, low carbon:nitrogen ratio, ammonia toxicity and even salinity. The present review summarizes and compares information concerning to anaerobic digestion of microalgal biomass and future directions for research. Besides, specific operational factors and potential inhibitory parameters of the process are analyzed and compared. Additionally, the paper covers the state or art concerning in methane production enhancement from algal biomass.     

Dual purpose microalgae-bacteria-based systems that treat wastewater and produce biodiesel and chemical products within a Biorefinery

Excess greenhouse gas emissions and the concomitant effect on global warming have become significant environmental, social and economic threats. In this context, the development of renewable, carbon-neutral and economically feasible biofuels is a driving force for innovation worldwide. A lot of effort has been put into developing biodiesel from microalgae. However, there are still a number of technological, market and policy barriers that are serious obstacles to the economic feasibility and competitiveness of such biofuels. Conversely, there are also a number of business opportunities if the production of such alternative biofuel becomes part of a larger integrated system following the Biorefinery strategy. In this case, other biofuels and chemical products of high added value are produced, contributing to an overall enhancement of the economic viability of the whole integrated system. Additionally, dual purpose microalgae-bacteria-based systems for treating wastewater and production of biofuels and chemical products significantly contribute to a substantial saving in the overall cost of microalgae biomass production. These types of systems could help to improve the competitiveness of biodiesel production from microalgae, according to some recent Life Cycle Analysis studies. Furthermore, they do not compete for fresh water resources for agricultural purposes and add value to treating the wastewater itself. This work reviews the most recent and relevant information about these types of dual purpose systems. Several aspects related to the treatment of municipal and animal wastewater with simultaneous recovery of microalgae with potential for biodiesel production are discussed. The use of pre-treated waste or anaerobic effluents from digested waste as nutrient additives for weak wastewater is reviewed. Isolation and screening of microalgae/cyanobacteria or their consortia from various wastewater streams, and studies related to population dynamics in mixed cultures, are highlighted as very relevant fields of research. The species selection may depend on various factors, such as the biomass and lipid productivity of each strain, the characteristics of the wastewater, the original habitat of the strain and the climatic conditions in the treatment plant, among others. Some alternative technologies aimed at harvesting biomass at a low cost, such as cell immobilization, biofilm formation, flocculation and bio-flocculation, are also reviewed. Finally, a Biorefinery design is presented that integrates the treatment of municipal wastewater with the recovery of oleaginous microalgae, together with the use of seawater supplemented with anaerobically digested piggery waste for cultivating Arthrospira (Spirulina) and producing biogas, biodiesel, hydrogen and other high added value products. Such strategies offer new opportunities for the cost-effective and competitive production of biofuels along with valuable non-fuel products.     

Biodiesel from microalgae beats bioethanol

Renewable biofuels are needed to displace petroleum-derived transport fuels, which contribute to global warming and are of limited availability. Biodiesel and bioethanol are the two potential renewable fuels that have attracted the most attention. As demonstrated here, biodiesel and bioethanol produced from agricultural crops using existing methods cannot sustainably replace fossil-based transport fuels, but there is an alternative. Biodiesel from microalgae seems to be the only renewable biofuel that has the potential to completely displace petroleum-derived transport fuels without adversely affecting supply of food and other crop products. Most productive oil crops, such as oil palm, do not come close to microalgae in being able to sustainably provide the necessary amounts of biodiesel. Similarly, bioethanol from sugarcane is no match for microalgal biodiesel.     

Validated numerical fluid simulation of a thin‐layer cascade photobioreactor in OpenFOAM

Recently, microalgae have been considered as a promising alternative for the production of biofuels from CO₂. For the efficient cultivation of these microalgae, several types of photobioreactors have been designed and Pilot scale photobioreactors have been used to assess the performance of these reactors. Therein the primarily investigated reactor type is the Raceway Pond. However, the less researched Thin‐Layer Cascade Photobioreactor (TLC) shows a high potential for efficient production processes. Unfortunately, for low‐value products like biofuels costs must be kept to a minimum for an economic operation. To facilitate this, 3D Computational Fluid Dynamic simulations can be employed to estimate performance of reactor variants e.g. with respect to power input and mixing. Since up to now little effort has been put into the modelling of TLC reactors, this report aims to present a simulation approach for these reactors types that allows simple adaptation to different geometric or operational boundary conditions. All models have been generated for a two‐phase mixture in OpenFOAM. To demonstrate its applicability, validation measurements with a physical unit have been performed and were compared to the simulation results. With errors in the order of 10 % a successful simulation of the reactor geometry could be proven.     

Biofuels from microalgae

Microalgae are a diverse group of prokaryotic and eukaryotic photosynthetic microorganisms that grow rapidly due to their simple structure. They can potentially be employed for the production of biofuels in an economically effective and environmentally sustainable manner. Microalgae have been investigated for the production of a number of different biofuels including biodiesel, bio-oil, bio-syngas, and bio-hydrogen. The production of these biofuels can be coupled with flue gas CO2 mitigation, wastewater treatment, and the production of high-value chemicals. Microalgal farming can also be carried out with seawater using marine microalgal species as the producers. Developments in microalgal cultivation and downstream processing (e.g., harvesting, drying, and thermochemical processing) are expected to further enhance the cost-effectiveness of the biofuel from microalgae strategy.     

Health benefits of microalgae and their microbiomes

Microalgae comprise a phylogenetically very diverse group of photosynthetic unicellular pro- and eukaryotic organisms growing in marine and other aquatic environments. While they are well explored for the generation of biofuels, their potential as a source of antimicrobial and prebiotic substances have recently received increasing interest. Within this framework, microalgae may offer solutions to the societal challenge we face, concerning the lack of antibiotics treating the growing level of antimicrobial resistant bacteria and fungi in clinical settings. While the vast majority of microalgae and their associated microbiota remain unstudied, they may be a fascinating and rewarding source for novel and more sustainable antimicrobials and alternative molecules and compounds. In this review, we present an overview of the current knowledge on health benefits of microalgae and their associated microbiota. Finally, we describe remaining issues and limitation, and suggest several promising research potentials that should be given attention.     

Potential of sponges and microalgae for marine biotechnology

Marine organisms can be used to produce several novel products that have applications in new medical technologies, in food and feed ingredients and as biofuels. In this paper two examples are described: the development of marine drugs from sponges and the use of microalgae to produce bulk chemicals and biofuels. Many sponges produce bioactive compounds with important potential applications as medical drugs. Recent developments in metagenomics, in the culturing of associated microorganisms from sponges and in the development of sponge cell-lines have the potential to solve the issue of supply, which is the main limitation for sponge exploitation. For the production of microalgal products at larger scales and the production of biofuels, major technological breakthroughs need to be realized to increase the product yield.     

Algae biofuels: versatility for the future of bioenergy

The world continues to increase its energy use, brought about by an expanding population and a desire for a greater standard of living. This energy use coupled with the realization of the impact of carbon dioxide on the climate, has led us to reanalyze the potential of plant-based biofuels. Of the potential sources of biofuels the most efficient producers of biomass are the photosynthetic microalgae and cyanobacteria. These versatile organisms can be used for the production of bioethanol, biodiesel, biohydrogen, and biogas. In fact, one of the most economic methods for algal biofuels production may be the combined biorefinery approach where multiple biofuels are produced from one biomass source.     

Algal biofuels

The world is facing energy crisis and environmental issues due to the depletion of fossil fuels and increasing CO₂ concentration in the atmosphere. Growing microalgae can contribute to practical solutions for these global problems because they can harvest solar energy and capture CO₂ by converting it into biofuel using photosynthesis. Microalgae are robust organisms capable of rapid growth under a variety of conditions including in open ponds or closed photobioreactors. Their reduced biomass compounds can be used as the feedstock for mass production of a variety of biofuels. As another advantage, their ability to accumulate or secrete biofuels can be controlled by changing their growth conditions or metabolic engineering. This review is aimed to highlight different forms of biofuels produced by microalgae and the approaches taken to improve their biofuel productivity. The costs for industrial-scale production of algal biofuels in open ponds or closed photobioreactors are analyzed. Different strategies for photoproduction of hydrogen by the hydrogenase enzyme of green algae are discussed. Algae are also good sources of biodiesel since some species can make large quantities of lipids as their biomass. The lipid contents for some of the best oil-producing strains of algae in optimized growth conditions are reviewed. The potential of microalgae for producing petroleum related chemicals or ready-make fuels such as bioethanol, triterpenic hydrocarbons, isobutyraldehyde, isobutanol, and isoprene from their biomass are also presented.     

Geographical Assessment of Microalgae Biofuels Potential Incorporating Resource Availability

Previous assessments of the economic feasibility and large-scale productivity of microalgae biofuels have not considered the impacts of land and carbon dioxide (CO₂) availability on the scalability of microalgae-based biofuels production. To accurately assess the near-term productivity potential of large-scale microalgae biofuel in the USA, a geographically realized growth model was used to simulate microalgae lipid yields based on meteorological data. The resulting lipid productivity potential of Nannochloropsis under large-scale cultivation is combined with land and CO₂ resource availability illustrating current geographically feasible production sites and corresponding productivity in the USA. Baseline results show that CO₂ transport constraints will limit US microalgae-based bio-oil production to 4 % of the 2030 Department of Energy (DOE) alternative fuel goal. The discussion focuses on synthesis of this large-scale productivity potential results including a sensitivity analysis to land and CO₂ resource assumptions, an evaluation of previous modeling efforts, and their assumptions regarding the transportation of CO₂, the feasibility of microalgae to meet DOE 2030 alternative fuel goals, and a comparison of the productivity potential in several key regions of the USA.     

莱茵衣藻中脂质代谢过程研究进展

微藻中脂质代谢产生的化合物,可用于生物燃料、营养品和生物药品的生产,因此具有重要的经济价值。脂质代谢贯穿微藻的全部生命过程,对微藻的生长发育和应对外界胁迫都具有重要意义。微藻与研究较清楚的真菌和陆地植物在脂质代谢过程方面具有相似性。当然,随着微藻脂质代谢相关功能基因逐渐被鉴定,人们发现微藻的脂质代谢也具有区别真菌和陆地植物的独特性,因此针对微藻脂质代谢过程的分析具有重要意义。莱茵衣藻是研究脂质代谢过程的模式生物,已经通过基因组、转录组、蛋白质组和代谢组等方法,对其质体、内质网和过氧化物酶体中进行的脂质合成和分解过程进行了研究。本文总结了近年来莱茵衣藻质体、内质网和过氧化物酶体中脂质代谢过程的研究成果,并进行综合阐述。     

Microalgal carbohydrates: an overview of the factors influencing carbohydrates production, and of main bioconversion technologies for production of biofuels

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.     

Genetic Engineering of Algae for Enhanced Biofuel Production

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 H₂ 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 H₂ 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.Interest in a variety of renewable biofuels has been rejuvenated due to the instability of petroleum fuel costs, the reality of peak oil in the near future, a reliance on unstable foreign petroleum resources, and the dangers of increasing atmospheric CO₂ levels. Photosynthetic algae, both microalgae and macroalgae (i.e., seaweeds), have been of considerable interest as a possible biofuel resource for decades (165). Several species have biomass production rates that can surpass those of terrestrial plants (41), and many eukaryotic microalgae have the ability to store significant amounts of energy-rich compounds, such as triacylglycerol (TAG) and starch, that can be utilized for the production of several distinct biofuels, including biodiesel and ethanol. It is believed that a large portion of crude oil is of microalgal origin, with diatoms being especially likely candidates, considering their lipid profiles and productivity (153). If ancient algae are responsible for creating substantial crude oil deposits, it is clear that investigation of the potential of living microalgae to produce biofuels should be a priority. Microalgae are especially attractive as a source of fuel from an environmental standpoint because they consume carbon dioxide and can be grown on marginal land, using waste or salt water (41). In addition, it may be possible to leverage the metabolic pathways of microalgae to produce a wide variety of biofuels (Fig. 1). In contrast to corn-based ethanol or soy/palm-based biodiesel, biofuels derived from microalgal feedstocks will not directly compete with the resources necessary for agricultural food production if inorganic constituents can be recycled and saltwater-based cultivation systems are developed.Open in a separate windowFig. 1.Microalgal metabolic pathways that can be leveraged for biofuel production. ER, endoplasmic reticulum.However, several technical barriers need to be overcome before microalgae can be used as an economically viable biofuel feedstock (139). These include developing low-energy methods to harvest microalgal cells, difficulties in consistently producing biomass at a large scale in highly variable outdoor conditions, the presence of invasive species in large-scale ponds, low light penetrance in dense microalgal cultures, the lack of cost-effective bioenergy carrier extraction techniques, and the potentially poor cold flow properties of most microalga-derived biodiesel. To advance the utilization of microalgae in biofuel production, it is important to engineer solutions to optimize the productivity of any microalgal cultivation system and undertake bioprospecting efforts to identify strains with as many desirable biofuel traits as possible. Over 40,000 species of algae have been described, and this is likely only a small fraction of the total number of available species (75). The U.S. Department of Energy''s Aquatic Species Program analyzed approximately 3,000 different microalgae for their potential to produce biofuels, and numerous additional species have subsequently been investigated (165). Although these efforts demonstrated that many species of microalgae have properties that are desirable for biofuel production, most have drawbacks that have prevented the emergence of an economically viable algal biofuel industry. It is postulated that a light-harvesting footprint of at least 20,000 square miles will be required to satisfy most of the current U.S. transportation fuel demand (41). Therefore, even modest improvements in photon conversion efficiencies will dramatically reduce the land area and cost required to produce biofuels. Consequently, continued bioprospecting efforts and the development and engineering of select microalgal strains are required to improve the yields of bioenergy carriers. Current commercial agriculture crops have been cultivated for thousands of years, with desired traits selected over time. It stands to reason that with microalgae, it would be beneficial to use genetic engineering in an attempt to bypass such a lengthy selection process. However, despite the recent advances in biotechnological approaches, the full potential of genetic engineering in some microalgal species, particularly diploid diatoms, can be fully realized only if conventional breeding methods become firmly established, thereby allowing useful traits or mutations to be easily combined (5, 24, 25). Since the topic of microalgal sexual breeding is beyond the scope of this review, we will instead focus on genetic engineering approaches that could be utilized in the industry''s efforts to improve microalgae as a source of biofuels.     

Microalgae bulk growth model with application to industrial scale systems

The scalability of microalgae growth systems is a primary research topic in anticipation of the commercialization of microalgae-based biofuels. To date, there is little published data on the productivity of microalgae in growth systems that are scalable to commercially viable footprints. To inform the development of more detailed assessments of industrial-scale microalgae biofuel processes, this paper presents the construction and validation of a model of microalgae biomass and lipid accumulation in an outdoor, industrial-scale photobioreactor. The model incorporates a time-resolved simulation of microalgae growth and lipid accumulation based on solar irradiation, species specific characteristics, and photobioreactor geometry. The model is validated with 9 weeks of growth data from an industrially-scaled outdoor photobioreactor. Discussion focuses on the sensitivity of the model input parameters, a comparison of predicted microalgae productivity to the literature, and an analysis of the implications of this more detailed growth model on microalgae biofuels lifecycle assessment studies.     

Isolation of high-lipid content strains of the marine microalga <Emphasis Type="Italic">Tetraselmis suecica</Emphasis> for biodiesel production by flow cytometry and single-cell sorting

Biodiesel from algae is considered an alternative for a third generation of biofuels. However, most microalgae are not lipogenic during fast growth periods, but high-lipid content occurs at resting stages. Microalgae biomass production for biodiesel needs continuous high volumetric and aerial yields and large amount of neutral lipid in the biomass. These requirements are similar to demanding a marathon runner to be obese. We show that by using cell sorting capabilities of flow cytometers, in combination with the lipid-soluble fluorescent dye Nile Red, we can isolate and select cells with a high and stable lipid content. In our study, we were able to select the equivalent of a stable “fat marathon runner” through three sorting events obtained from wild populations of Tetraselmis suecica.