Co-Cultivation of Fungal and Microalgal Cells as an Efficient System for Harvesting Microalgal Cells,Lipid Production and Wastewater Treatment

The challenges which the large scale microalgal industry is facing are associated with the high cost of key operations such as harvesting, nutrient supply and oil extraction. The high-energy input for harvesting makes current commercial microalgal biodiesel production economically unfeasible and can account for up to 50% of the total cost of biofuel production. Co-cultivation of fungal and microalgal cells is getting increasing attention because of high efficiency of bio-flocculation of microalgal cells with no requirement for added chemicals and low energy inputs. Moreover, some fungal and microalgal strains are well known for their exceptional ability to purify wastewater, generating biomass that represents a renewable and sustainable feedstock for biofuel production. We have screened the flocculation efficiency of the filamentous fungus A. fumigatus against 11 microalgae representing freshwater, marine, small (5 µm), large (over 300 µm), heterotrophic, photoautotrophic, motile and non-motile strains. Some of the strains are commercially used for biofuel production. Lipid production and composition were analysed in fungal-algal pellets grown on media containing alternative carbon, nitrogen and phosphorus sources contained in wheat straw and swine wastewater, respectively. Co-cultivation of algae and A. fumigatus cells showed additive and synergistic effects on biomass production, lipid yield and wastewater bioremediation efficiency. Analysis of fungal-algal pellet''s fatty acids composition suggested that it can be tailored and optimised through co-cultivating different algae and fungi without the need for genetic modification.     

Effects of bacterial communities on biofuel-producing microalgae: stimulation,inhibition and harvesting

Despite the great interest in microalgae as a potential source of biofuel to substitute for fossil fuels, little information is available on the effects of bacterial symbionts in mass algal cultivation systems. The bacterial communities associated with microalgae are a crucial factor in the process of microalgal biomass and lipid production and may stimulate or inhibit growth of biofuel-producing microalgae. In addition, we discuss here the potential use of bacteria to harvest biofuel-producing microalgae. We propose that aggregation of microalgae by bacteria to achieve >90% reductions in volume followed by centrifugation could be an economic approach for harvesting of biofuel-producing microalgae. Our aims in this review are to promote understanding of the effects of bacterial communities on microalgae and draw attention to the importance of this topic in the microalgal biofuel field.     

Strategies to enhance production of microalgal biomass and lipids for biofuel feedstock

ABSTRACT

Microalgae have enormous potential as feedstock for biofuel production compared with other sources, due to their high areal productivity, relatively low environmental impact, and low impact on food security. However, high production costs are the major limitation for commercialization of algal biofuels. Strategies to maximize biomass and lipid production are crucial for improving the economics of using microalgae for biofuels. Selection of suitable algal strains, preferably from indigenous habitats, and further improvement of those ‘platform strains’ using mutagenesis and genetic engineering approaches are desirable. Conventional approaches to improve biomass and lipid productivity of microalgae mainly involve manipulation of nutritional (e.g. nitrogen and phosphorus) and environmental (e.g. temperature, light and salinity) factors. Approaches such as the addition of phytohormones, genetic and metabolic engineering, and co-cultivation of microalgae with yeasts and bacteria are more recent strategies to enhance biomass and lipid productivity of microalgae. Improvement in culture systems and the use of a hybrid system (i.e. a combination of open ponds and photobioreactors) is another strategy to optimize algal biomass and lipid production. In addition, the use of low-cost substrates such as agri-industrial wastewater for the cultivation of microalgae will be a smart strategy to reduce production costs. Such systems not only generate high algal biomass and lipid productivity, but are also useful for bioremediation of wastewater and bioremoval of waste CO₂. The aim of this review is to highlight the advances in the use of various strategies to enhance production of algal biomass and lipids for biofuel feedstock.     

Molecular Identification and Comparative Evaluation of Tropical Marine Microalgae for Biodiesel Production

Marine microalgae have emerged as important feedstock for liquid biofuel production. The identification of lipid-rich native microalgal species with high growth rate and optimal fatty acid profile and biodiesel properties is the most challenging step in microalgae-based biodiesel production. In this study, attempts have been made to bio-prospect the biodiesel production potential of marine and brackish water microalgal isolates from the west coast of India. A total of 14 microalgal species were isolated, identified using specific molecular markers and based on the lipid content; seven species with total lipid content above 20% of dry cell weight were selected for assessing biodiesel production potential in terms of lipid and biomass productivities, nile red fluorescence, fatty acid profile and biodiesel properties. On comparative analysis, the diatoms were proven to be promising based on the overall desirable properties for biodiesel production. The most potential strain Navicula phyllepta MACC8 with a total lipid content of 26.54 % of dry weight of biomass, the highest growth rate (0.58 day^?1) and lipid and biomass productivities of 114 and 431 mgL^?1 day^?1, respectively, was rich in fatty acids mainly of C16:0, C16:1 and C18:0 in the neutral lipid fraction, the most favoured fatty acids for ideal biodiesel properties. The biodiesel properties met the requirements of fuel quality standards based on empirical estimation. The marine diatoms hold a great promise as feedstock for large-scale biodiesel production along with valuable by-products in a biorefinery perspective, after augmenting lipid and biomass production through biochemical and genetic engineering approaches.     

Metabolomics integrated with transcriptomics and proteomics: Evaluation of systems reaction to nitrogen deficiency stress in microalgae

Microalgae have higher productivity of biomass than the conventional crops of fuel and are therefore, considered a potential biofuel source. Lipid, an important precursor of biodiesel, can be overproduced in microalgae by nitrogen deprivation. During nitrogen deficiency, radicals are overproduced, and the antioxidant levels are insufficient to counteract the radicals. Thus, the increase in cellular oxidative stress level, consequently acts as a stimulus for lipid accumulation. Lipid accumulation requires an excess of acetyl CoA and NADPH that is made possible by the following mechanism. Glycolysis upregulation overproduces pyruvate, which could be further transformed into acetyl CoA by the pyruvate dehydrogenase complex; while the upregulation of the oxidative pentose phosphate cycle generates a high amount of NADPH. In addition to lipid overproduction, the lack of nitrogen often causes the accumulation of carbohydrates in selected species of microalgae, which could be used to generate biogas and bioethanol from the defatted biomass. By providing details on the differential regulation of the biochemical pathways leading to lipid and carbohydrate accumulation in nitrogen starved microalgae, the review opens up new possibilities in the microalgal biofuel production.     

Integration of biological waste conversion and wastewater treatment plants by microalgae cultivation

In this study, growth performance and lipid content of two microalgae species Neochloris oleoabundans and Chlorella vulgaris are monitored by using three different types of sludge waste feedstocks obtained from the water treatment plants located in Bedonia, Borgotaro and Fornovo (Montagna2000 Spa, Province of Parma, Italy). The sludge waste is optimized in order to achieve microalgal growth media and dispose of the sewage sludge produced at the wastewater treatment facilities. Both photoautotrophic and heterotrophic growth conditions are applied to the microalgal cultivations. The growth parameters of microalgae strains such as cell concentration, growth rate, optical density, cell biovolume, photosynthetic pigments and lipid contents are monitored. The amounts of total dried lipid biomass, obtained by the biological conversion of the wet sludge waste, are determined. Lipid production of microalgal cells grown in the medium optimized from sludge waste from the Fornovo site provides the highest amount of microalgal lipid content for N. oleoabundans and C. vulgaris photoautotrophic cultivations, while sludge waste from the Bedonia site provides for N. oleoabundans heterotrophic cultivation.     

Microalgae–bacteria symbiosis in microalgal growth and biofuel production: a review

Photosynthetic microalgae can capture solar energy and convert it to bioenergy and biochemical products. In nature or industrial processes, microalgae live together with bacterial communities and may maintain symbiotic relationships. In general interactions, microalgae exude dissolved organic carbon that becomes available to bacteria. In return, the bacteria remineralize sulphur, nitrogen and phosphorous to support the further growth of microalgae. In specific interactions, heterotrophic bacteria supply B vitamins as organic cofactors or produce siderophores to bind iron, which could be utilized by microalgae, while the algae supply fixed carbon to the bacteria in return. In this review, we focus on mutualistic relationship between microalgae and bacteria, summarizing recent studies on the mechanisms involved in microalgae–bacteria symbiosis. Symbiotic bacteria on promoting microalgal growth are described and the relevance of microalgae–bacteria interactions for biofuel production processes is discussed. Symbiotic microalgae–bacteria consortia could be utilized to improve microalgal biomass production and to enrich the biomass with valuable chemical and energy compounds. The suitable control of such biological interactions between microalgae and bacteria will help to improve the microalgae-based biomass and biofuel production in the future.     

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.     

Bioflocculation as an innovative harvesting strategy for microalgae

Microalgae are a promising new source of biomass for the production of third generation biofuels but, so far, the majority of microalgal biomass has been used for high-value applications. New low-cost technologies are needed to make the production and processing of microalgae economically feasible for low-value applications. A major challenge lies in the harvesting of microalgae, which requires a cost-efficient separation technology. Flocculation, especially bioflocculation, is an attractive low-cost separation technology. Various new bioflocculation strategies have been claimed to generate major advances in cost-efficient harvesting. Here, we review the recent advances in bioflocculation based on algal–bacterial, algal–fungal, or algal–algal interactions within the framework of microalgae biomass harvesting for biofuel production. We also discuss recent advances using infochemicals and genetic engineering for the induction of bioflocculation.     

Functional group richness: implications of biodiversity for light use and lipid yield in microalgae

Currently, very few studies address the relationship between diversity and biomass/lipid production in primary producer communities for biofuel production. Basic studies on the growth of microalgal communities, however, provide evidence of a positive relationship between diversity and biomass production. Recent studies have also shown that positive diversity–productivity relationships are related to an increase in the efficiency of light use by diverse microalgal communities. Here, we hypothesize that there is a relationship between diversity, light use, and microalgal lipid production in phytoplankton communities. Microalgae from all major freshwater algal groups were cultivated in treatments that differed in species richness and functional group richness. Polycultures with high functional group richness showed more efficient light use and higher algal lipid content with increasing species richness. There was a clear correlation between light use and lipid production in functionally diverse communities. Hence, a powerful and cost‐effective way to improve biofuel production might be accomplished by incorporating diversity related, resource‐use‐dynamics into algal biomass production.     

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.     

生物柴油原料资源高油脂微藻的开发利用

生物柴油作为化石能源的替代燃料已在国际上得到广泛应用。至今生物柴油的原料主要来自油料植物, 但与农作物争地的情况以及较高的原料成本限制了生物柴油的进一步推广。微藻作为高光合生物有其特殊的原料成本优势, 微藻的脂类含量最高可达细胞干重的80%。利用生物技术改良微藻, 获得的高油脂基因工程微藻经规模养殖, 可大大降低生物柴油原料成本。介绍了国内外生物柴油的应用现状, 阐述了微藻作为生物柴油原料的优势, 对基因工程技术调控微藻脂类代谢途径的研究进展, 以及在构建工程微藻中面临的问题和应采取的对策进行了综述和展望。     

Effect of nutrients on growth and lipid accumulation in the green algae Dunaliella tertiolecta

Production of biofuel from algae is dependent on the microalgal biomass production rate and lipid content. Both biomass production and lipid accumulation are limited by several factors, of which nutrients play a key role. In this research, the marine microalgae Dunaliella tertiolecta was used as a model organism and a profile of its nutritional requirements was determined. Inorganic phosphate PO4(3-) and trace elements: cobalt (Co2+), iron (Fe3+), molybdenum (Mo2+) and manganese (Mn2+) were identified as required for algae optimum growth. Inorganic nitrogen in the form of nitrate NO3- instead of ammonium (NH4+) was required for maximal biomass production. Lipids accumulated under nitrogen starvation growth condition and this was time-dependent. Results of this research can be applied to maximize production of microalgal lipids in optimally designed photobioreactors.     

Biodiesel production with microalgae as feedstock: from strains to biodiesel

Due to negative environmental influence and limited availability, petroleum-derived fuels need to be replaced by renewable biofuels. Biodiesel has attracted intensive attention as an important biofuel. Microalgae have numerous advantages for biodiesel production over many terrestrial plants. There are a series of consecutive processes for biodiesel production with microalgae as feedstock, including selection of adequate microalgal strains, mass culture, cell harvesting, oil extraction and transesterification. To reduce the overall production cost, technology development and process optimization are necessary. Genetic engineering also plays an important role in manipulating lipid biosynthesis in microalgae. Many approaches, such as sequestering carbon dioxide from industrial plants for the carbon source, using wastewater for the nutrient supply, and maximizing the values of by-products, have shown a potential for cost reduction. This review provides a brief overview of the process of biodiesel production with microalgae as feedstock. The methods associated with this process (e.g. lipid determination, mass culture, oil extraction) are also compared and discussed.     

湛江沿海海洋微藻及其多糖、脂类和蛋白藻株多样性研究

从硇洲岛和徐闻珊瑚礁自然保护区潮间带采集的海水和沉积物样品中分离培养海洋微藻, 筛选其中富含多糖、脂类或蛋白质的藻株。采用形态学观察、18S rDNA序列比较及其系统发育分析法, 对分离培养的海洋微藻及其富含多糖、脂类或蛋白质的藻株进行分类鉴定和生物多样性分析。分离、培养、鉴定并储藏了189株海洋微藻, 归属于65个种, 分布于硅藻门(Bacillariophyta)、绿藻门(Chlorophyta)、定鞭藻门(Haptophyta)和红藻门(Rhodophyta)的9纲、25目、30个科、38个属; 其中多糖含量较高的46株海洋微藻, 分布于25个种, 20个属; 脂类含量较高的46株海洋微藻, 分布于32个种, 15个属; 蛋白含量较高的46株海洋微藻, 分布于28个种, 18个属。结果表明硇洲岛和徐闻珊瑚礁自然保护区潮间带可培养的海洋微藻及其富含多糖、脂类和蛋白质藻株的物种多样性丰富, 在新型药物、活性天然产物、功能食品和饲料及其添加剂的发掘等方面具有良好前景。     

Methods of downstream processing for the production of biodiesel from microalgae

Despite receiving increasing attention during the last few decades, the production of microalgal biofuels is not yet sufficiently cost-effective to compete with that of petroleum-based conventional fuels. Among the steps required for the production of microalgal biofuels, the harvest of the microalgal biomass and the extraction of lipids from microalgae are two of the most expensive. In this review article, we surveyed a substantial amount of previous work in microalgal harvesting and lipid extraction to highlight recent progress in these areas. We also discuss new developments in the biodiesel conversion technology due to the importance of the connectivity of this step with the lipid extraction process. Furthermore, we propose possible future directions for technological or process improvements that will directly affect the final production costs of microalgal biomass-based biofuels.     

Perspectives on microalgal CO₂-emission mitigation systems--a review

The problem of climate change arising mainly from CO? emission is currently a critical environmental issue. Biofixation using microalgae has recently become an attractive approach to CO? capture and recycling with additional benefits of downstream utilization and applications of the resulting microalgal biomass. This review summarizes the history and strategies of microalgal mitigation of CO? emissions, photobioreactor systems used to cultivate microalgae for CO? fixation, current microalgae harvesting methods, as well as applications of valuable by-products. It is of importance to select appropriate microalgal species to achieve an efficient and economically feasible CO?-emission mitigation process. The desired microalgae species should have a high growth rate, high CO? fixation ability, low contamination risk, low operation cost, be easy to harvest and rich in valuable components in their biomass.     

Mixotrophic and photoautotrophic cultivation of 14 microalgae isolates from Saskatchewan, Canada: potential applications for wastewater remediation for biofuel production

Northern regions are generally viewed as unsuitable for microalgal biofuel production due to unfavorable climate and solar insolation levels. However, these conditions can potentially be mitigated by coupling microalgal cultivation to industrial processes such as wastewater treatment. In this study, we have examined the biomass and lipid productivity characteristics of 14 microalgae isolates (Chlorophyta) from the Canadian province of Saskatchewan. Under both photoautotrophic and mixotrophic cultivation, a distinct linear trend was observed between biomass and lipid productivities in the 14 SK isolates. The most productive strain under cultivation in TAP media was Scenedesmus sp.-AMDD which displayed rates of biomass and fatty acid productivities of 80 and 30.7?mg?L^?1?day^?1, respectively. The most productive strain in B3NV media was Chlamydomonas debaryana-AMLs1b which displayed rates of biomass and fatty acid productivities of 51.7 and 5.9?mg?L^?1?day^?1, respectively. In 11 of the isolates tested, secondary municipal wastewater (MCWW) supported rates of biomass productivity between 21 and 33?mg?L^?1?day^?1 with Scenedesmus sp.-AMDD being the most productive. Three strains, Chlamydomonas debaryana-AMB1, Chlorella sorokiniana-RBD8 and Micractinium sp.-RB1b, showed large increases in biomass productivity when cultivated mixotrophically in MCWW supplemented with glycerol. High relative oleic acid content was detected in 10 of the 14 isolates when grown mixotrophically in media supplemented with acetate. There was no detectable effect on the fatty acid profiles in cells cultivated mixotrophically in glycerol-supplemented MCWW. These data indicate that biomass and lipid productivities are boosted by mixotrophic cultivation. Exploiting this response in municipal wastewater is a promising strategy for the production of environmentally sustainable biofuels.     

Microalgae engineering toolbox: Selectable and screenable markers

Engineering microalgae has opened a new era for plant biologists and biotechnologists. Microalgae had been proved as a promising candidate for the production of biopharmaceuticals, nutraceuticals, antioxidants, antimicrobial and antiviral compounds, in dyeing and food industry as well for biofuel production. Genetic transformation of some important microalgae has been successful, but several other potential microalgae species still need scientific attention. The success of the genetic transformation depends mainly on the utilization of the selectable and screenable markers. Like for other higher crop plants, several useful markers have been reported for microalgae transformation. In this follow-up, we compared different marker genes for genetic engineering of approximately all the industrially important microalgae. We have discussed the expression host, the targeted genome, appropriate selection agent, as well as the transformation method. Genetic transformation is an expensive and labor intensive process and this review will aid to shorten the time span by providing a database of appropriate markers for microalgae research which could serve as a guide for those involved in the genetic engineering of microalgae.     

In situ solvent recovery by using hydrophobic/oleophilic filter during wet lipid extraction from microalgae

Bioprocess and Biosystems Engineering - While lipid extraction from wet microalgae has attracted attention as an economical method for microalgal biofuel production, few studies have focused the...