微藻异养高密度培养研究进展与发展趋势*

微藻细胞可以积累大量油脂、蛋白质、多糖、色素、不饱和脂肪酸等物质,在能源、食品、饵料、保健品及药品等行业有巨大的应用价值。然而,微藻在传统光自养模式下很难实现高密度培养来大量生产这些重要的物质,进而限制了微藻的实际应用。相反,微藻在异养模式下生长速度快、生物质浓度高,可以短时间内获得大量微藻生物质。因此,异养高密度培养微藻具备大规模、高效率培养微藻生产目标产物的巨大潜力。阐述微藻异养培养的优缺点及相应技术难点的解决思路、影响微藻异养生长及目标产物积累的主要营养因子和环境因子、微藻异养高密度培养的方式及微藻异养高密度培养的当前发展水平。结合文献报道分析微藻异养高密度培养的四个具有极大发展潜力的发展方向,以期更好地利用异养模式来高效率、低成本培养微藻生产大量目标产物,满足上述多个行业对微藻原材料的巨大需求,从而加速微藻产业的发展。     

Anaerobic digestion of microalgal biomass for bioenergy production,removal of nutrients and microcystin: current status

Using renewable microalgal biomass as active feedstocks for biofuels and bioproducts is explored to substitute petroleum-based fuels and chemicals. In the last few years, the importance of microalgae biomass has been realized as a renewable feedstock due to several positive attributes associated with it. Biorefinery via anaerobic digestion (AD) of microalgal biomass is a promising and sustainable method to produce value-added chemicals, edible products and biofuels. Microalgal biomass pretreatment is a significant process to enhance methane production by AD. Findings on the AD microbial community’s variety and organization can give novel in turn on digester steadiness and presentation. This review presents a vital study of the existing facts on the AD microbial community and AD production. Co-digestion of microalgal biomass with different co-substrates was used in AD to enhance biogas production, and the process was economically viable with improved biodegradability. Microcystins, which are produced by toxic cyanobacterial blooms, create a severe hazard to environmental health. Anaerobic biodegradation is an effective method to degrade the microcystins and convert into nontoxic products. However, for the cost-effective conversion of biomass to energy and other beneficial byproducts, additional highly developed research is still required for large-scale AD of microalgal biomass.     

Extraction of oil from microalgae for biodiesel production: A review

The rapid increase of CO(2) concentration in the atmosphere combined with depleted supplies of fossil fuels has led to an increased commercial interest in renewable fuels. Due to their high biomass productivity, rapid lipid accumulation, and ability to survive in saline water, microalgae have been identified as promising feedstocks for industrial-scale production of carbon-neutral biodiesel. This study examines the principles involved in lipid extraction from microalgal cells, a crucial downstream processing step in the production of microalgal biodiesel. We analyze the different technological options currently available for laboratory-scale microalgal lipid extraction, with a primary focus on the prospect of organic solvent and supercritical fluid extraction. The study also provides an assessment of recent breakthroughs in this rapidly developing field and reports on the suitability of microalgal lipid compositions for biodiesel conversion.     

Active extracellular substances of Bacillus thuringiensis ITRI‐G1 induce microalgae self‐disruption for microalgal biofuel

Microalgal cultures are a clean and sustainable means to use solar energy for CO₂ fixation and fuel production. Microalgae grow efficiently and are rich in oil, but recovering that oil is typically expensive and consumes much energy. Therefore, effective and low‐cost techniques for microalgal disruption and oil or lipid extraction are required by the algal biofuel industry. This study introduces a novel technique that uses active extracellular substances to induce microalgal cell disruption. A bacterium indigenous to Taiwan, Bacillus thuringiensis, was used to produce the active extracellular substances, which were volatile compounds with high thermal stability. Approximately 74% of fresh microalgal cells were disrupted after a 12‐h treatment with the active extracellular substances. Algal lipid extraction efficiency was improved and the oil extraction time was decreased by approximately 37.5% compared with the control treatment. The substances effectively disrupted fresh microalgal cells but not dehydrated microalgal cells. An analysis of microalgal DNA from fresh cells after disruption treatment demonstrated typical DNA laddering, indicating that disruption may have resulted from programmed cell death. This study revealed that biological treatments are environmentally friendly methods for increasing microalgal lipid extraction efficiency, and introduced a microalgal cell self‐disruption mechanism.     

Tris not only controls the pH in microalgal cultures,but also feeds bacteria

Tris (Tris(hydroxymethyl)amino methane), a compound often used as a buffer in microalgal culture media, sustains active bacterial growth in non-axenic microalgal cultures when sodium phosphate is present. The low pH levels caused by bacterial growth and probably the depletion of phosphorus in the medium caused the collapse ofPhaeodactylum tricornutum cultures resulting in a reduction of microalgal growth from 32 x 10⁶ to 1.1 x 10⁶ cells ml^–1. This emphasizes the need for care when interpreting the results of non-axenic microalgae cultures in which Tris or other organic buffer is added.     

Integration of microalgae cultivation with industrial waste remediation for biofuel and bioenergy production: opportunities and limitations

There is currently a renewed interest in developing microalgae as a source of renewable energy and fuel. Microalgae hold great potential as a source of biomass for the production of energy and fungible liquid transportation fuels. However, the technologies required for large-scale cultivation, processing, and conversion of microalgal biomass to energy products are underdeveloped. Microalgae offer several advantages over traditional 'first-generation' biofuels crops like corn: these include superior biomass productivity, the ability to grow on poor-quality land unsuitable for agriculture, and the potential for sustainable growth by extracting macro- and micronutrients from wastewater and industrial flue-stack emissions. Integrating microalgal cultivation with municipal wastewater treatment and industrial CO(2) emissions from coal-fired power plants is a potential strategy to produce large quantities of biomass, and represents an opportunity to develop, test, and optimize the necessary technologies to make microalgal biofuels more cost-effective and efficient. However, many constraints on the eventual deployment of this technology must be taken into consideration and mitigating strategies developed before large scale microalgal cultivation can become a reality. As a strategy for CO(2) biomitigation from industrial point source emitters, microalgal cultivation can be limited by the availability of land, light, and other nutrients like N and P. Effective removal of N and P from municipal wastewater is limited by the processing capacity of available microalgal cultivation systems. Strategies to mitigate against the constraints are discussed.     

MICROALGAL BIOTRANSFORMATION OF STEROIDS1

Microbial biotransformation of steroids is not a new concept, but most studies in this field have focused on fungal and bacterial systems. Microalgae, despite their photosynthetic ability and immense biodiversity, have not received much attention in this aspect until recently. Since the publication of the first article on microalgal biotransformation of steroids about 20 years ago, there have been many reports describing different modifications, including hydroxylation, reduction, side‐chain degradation, and isomerization introduced by these microorganisms on estrane, androstane, and pregnane derivatives. On the other hand, the development of new large‐scale cultivation systems, the adaptation of existing fermentation techniques to microalgae, and the introduction of microalgal genetic manipulation methods have made these organisms promising candidates for a wide range of biotechnological processes, including biotransformations. In this review, we have summarized the steroid transformation patterns of several microalgal strains and present a perspective of the future trends in microalgal biotechnology, including the possibility of adapting relatively new techniques, such as organic media catalysis and cell immobilization, to this specific field.     

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。     

Deciphering interactions between the marine dinoflagellate Prorocentrum lima and the fungus Aspergillus pseudoglaucus

The comprehension of microbial interactions is one of the key challenges in marine microbial ecology. This study focused on exploring chemical interactions between the toxic dinoflagellate Prorocentrum lima and a filamentous fungal species, Aspergillus pseudoglaucus, which has been isolated from the microalgal culture. Such interspecies interactions are expected to occur even though they were rarely studied. Here, a co-culture system was designed in a dedicated microscale marine-like condition. This system allowed to explore microalgal–fungal physical and metabolic interactions in presence and absence of the bacterial consortium. Microscopic observation showed an unusual physical contact between the fungal mycelium and dinoflagellate cells. To delineate specialized metabolome alterations during microalgal–fungal co-culture metabolomes were monitored by high-performance liquid chromatography coupled to high-resolution mass spectrometry. In-depth multivariate statistical analysis using dedicated approaches highlighted (1) the metabolic alterations associated with microalgal–fungal co-culture, and (2) the impact of associated bacteria in microalgal metabolome response to fungal interaction. Unfortunately, only a very low number of highlighted features were fully characterized. However, an up-regulation of the dinoflagellate toxins okadaic acid and dinophysistoxin 1 was observed during co-culture in supernatants. Such results highlight the importance to consider microalgal–fungal interactions in the study of parameters regulating toxin production.     

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.     

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.     

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.     

A simple and rapid method for separation and isolation of marine algal species from naturally evolved populations

To improve the study of mixed microalgal populations, three naturally evolved marine microalgal cultures were subjected to a light crushing mechanical treatment using a silicon spatula coupled with zymolyase treatment at four concentrations: 5, 10, 20 and 25 U/ml, for 15, 30, 45 and 60 min before being observed under a microscope. The enzyme concentration of 20 U/ml after 45 min reduces the size of macroscopic microalgal aggregates and improves the microscopic observation of the different microalgal species comprising the population. There was no improvement using the higher enzyme concentration. This paper proposes a new approach to the study of naturally evolved microalgal populations which is useful for distinguishing the morphology of the different species present in the population and allowing for the identification by classical keys, and also to obtain a pure culture from an inoculum of mixed species by using a micromanipulator for cell counting.     

Manipulation of the microalgal chloroplast by genetic engineering for biotechnological utilization as a green biofactory

The chloroplast is an essential organelle in microalgae for conducting photosynthesis, thus enabling the photoautotrophic growth of microalgae. In addition to photosynthesis, the chloroplast is capable of various biochemical processes for the synthesis of proteins, lipids, carbohydrates, and terpenoids. Due to these attractive characteristics, there has been increasing interest in the biotechnological utilization of microalgal chloroplast as a sustainable alternative to the conventional production platforms used in industrial biotechnology. Since the first demonstration of microalgal chloroplast transformation, significant development has occurred over recent decades in the manipulation of microalgal chloroplasts through genetic engineering. In the present review, we describe the advantages of the microalgal chloroplast as a production platform for various bioproducts, including recombinant proteins and high-value metabolites, features of chloroplast genetic systems, and the development of transformation methods, which represent important factors for gene expression in the chloroplast. Furthermore, we address the expression of various recombinant proteins in the microalgal chloroplast through genetic engineering, including reporters, biopharmaceutical proteins, and industrial enzymes. Finally, we present many efforts and achievements in the production of high-value metabolites in the microalgal chloroplast through metabolic engineering. Based on these efforts and advances, the microalgal chloroplast represents an economically viable and sustainable platform for biotechnological applications in the near future.     

Growth Rate of the Microalga Tetraselmis suecica Changes the Biochemical Composition of Artemia Species

The impact of different microalgal semicontinuous cultures on growth and biochemical composition in the next link of the food chain was tested using the filter feeder Artemia species as a model. The marine microalga Tetraselmis suecica was cultured semicontinuously with renewal rates between 10% and 50% and used to feed Artemia. Microalgal cultures maintained with a low renewal rate that had biochemical composition similar to that of the stationary-phase cultures commonly used in aquaculture produced poor growth and survival and low food-conversion efficiency compared to cultures maintained with a high renewal rate. Changes in the renewal rate in microalgal cultures also resulted in important changes in the gross biochemical composition of the filter feeder. The gross biochemical composition of the Artemia resembled that of the microalgae used as food except for total lipid content. The percentage of protein in the organic fraction of Artemia increased from 45% to 65% of the organic weight with increasing renewal rates in the microalgal cultures, while the carbohydrate percentage decreased under the same conditions. Higher renewal rates resulted in higher lipid percentages in the microalga, but in Artemia the percentage of lipids decreased from 19% of the organic weight with a renewal rate of 10%, to 13% with a renewal rate of 50%. The percentage of all polyunsaturated fatty acids in Artemia, including 20:5n-3, increased slightly with increasing renewal rates in the microalgal cultures. Results emphasize the importance of controlling microalgal nutritional value for the success of aquaculture food chains in which filter feeders are involved. Received October 15, 2000; accepted December 29, 2000.     

Perspectives on microalgal CO2-emission mitigation systems — A review

The problem of climate change arising mainly from CO₂ emission is currently a critical environmental issue. Biofixation using microalgae has recently become an attractive approach to CO₂ 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 CO₂ emissions, photobioreactor systems used to cultivate microalgae for CO₂ 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 CO₂-emission mitigation process. The desired microalgae species should have a high growth rate, high CO₂ fixation ability, low contamination risk, low operation cost, be easy to harvest and rich in valuable components in their biomass.     

Commercial development of microalgal biotechnology: from the test tube to the marketplace

While humans have taken limited advantage of natural populations of microalgae for centuries (Nostoc in Asia and Spirulina in Africa and North America for sustenance), it is only recently that we have come to realize the potential of microalgal biotechnology. Microalgal biotechnology has the potential to produce a vast array of products including foodstuffs, industrial chemicals, compounds with therapeutic applications and bioremediation solutions from a virtually untapped source. From an industrial (i.e. commercial) perspective, the goal of microalgal biotechnology is to make money by developing marketable products. For such a business to succeed the following steps must be taken: identify a desirable metabolite and a microalga that produces and accumulates the desired metabolite, establish a large-scale production process for the desired metabolite, and market the desired metabolite. So far, the commercial achievements of microalgal biotechnology have been modest. Microalgae that produce dozens of desirable metabolites have been identified. Aided by high throughput screening technology even more leads will become available. However, the successes in large-scale production and product marketing have been few. We will discuss those achievements and difficulties from the industrial point of view by considering examples from industry, specially our own experience at Mera Pharmaceuticals.     

Biofouling in photobioreactors for marine microalgae

The economic and/or energetic feasibility of processes based on using microalgae biomass requires an efficient cultivation system. In photobioreactors (PBRs), the adhesion of microalgae to the transparent PBR surfaces leads to biofouling and reduces the solar radiation penetrating the PBR. Light reduction within the PBR decreases biomass productivity and, therefore, the photosynthetic efficiency of the cultivation system. Additionally, PBR biofouling leads to a series of further undesirable events including changes in cell pigmentation, culture degradation, and contamination by invasive microorganisms; all of which can result in the cultivation process having to be stopped. Designing PBR surfaces with proper materials, functional groups or surface coatings, to prevent microalgal adhesion is essential for solving the biofouling problem. Such a significant advance in microalgal biotechnology would enable extended operational periods at high productivity and reduce maintenance costs. In this paper, we review the few systematic studies performed so far and applied the existing thermodynamic and colloidal theories for microbial biofouling formation in order to understand microalgal adhesion on PBR surfaces and the microalgae–microalgae cell interactions. Their relationship to the physicochemical properties of the solid PBR surface, the microalgae cell surfaces, and the ionic strength of the culture medium is discussed. The suitability and the applicability of such theories are reviewed. To this end, an example of biofouling formation on a commercial glass surface is presented for the marine microalgae Nannochloropsis gaditana. It highlights the adhesion dynamics and the inaccuracies of the process and the need for further refinement of previous theories so as to apply them to flowing systems, such as is the case for PBRs used to culture microalgae.     

Multi-parameter flow cytometry as a tool to monitor heterotrophic microalgal batch fermentations for oil production towards biodiesel

Multi-parameter flow cytometry was used to monitor cell intrinsic light scatter, viability, and lipid content of Chlorella protothecoides cells grown in shake flasks. Changes in the right angle light scatter (RALS) and forward angle light scatter (FALS) were detected during the microalgal growth, which were attributed to the different microalgal cell cycle stages. The proportion of cells not stained with PI (cells with intact cytoplasmic membrane) was high (> 90%) during the microalgal growth, even in the latter stationary phase, suggesting that the microalgal cells built-up storage materials which allowed them to survive under nutrient starvation, maintaining their cytoplasmic membranes intact. A high correlation between the Nile Red fluorescence intensity measured by flow cytometry and total lipid content assayed by the traditional lipid extraction method was found for this microalga, making this method a suitable and quick technique for the screening of microalgal strains for lipid production, optimization of biofuel production bioprocesses, and scale-up studies. The highest oil content (∼28% w/w dry cell weight, estimated by flow cytometry) was observed in the latter stationary phase. In addition, C. protothecoides oil also depicted the adequate fatty acid methyl ester composition for biodiesel purposes at this growth phase, suggesting that the microalgal oil produced during the latter stationary phase could be an adequate substitute for diesel fuel. Medium growth optimization for enhancement of microalgal oil production is now in progress, using the multi-parameter approach.     

The sea-ice algal community of seasonal pack ice in the southwestern Kara Sea in late winter

The composition and abundance of ice algae have been studied in a 64-cm core of seasonal pack ice taken in the southwestern Kara Sea in February 1996. The cell numbers, biomass and taxonomic diversity of the ice microalgal assemblages were rather low, and there was a predominance of diatoms. The comparison of its composition with that of the pelagic microalgal community demonstrated a great difference between them. The ecological function of the seasonal ice habitat as a depository for seeding material of the early-spring pelagic diatom is suggested. Accepted: 7 August 2000