Ultrahigh-cell-density heterotrophic cultivation of the unicellular green microalga Scenedesmus acuminatus and application of the cells to photoautotrophic culture enhance biomass and lipid production

Although production of biodiesels from microalgae is proved to be technically feasible, a commercially viable system has yet to emerge. High-cell-density fermentation of microalgae can be coupled with photoautotrophic cultivation to produce oils. In this study, by optimizing culturing conditions and employing a sophisticated substrate feed control strategy, ultrahigh-cell-density of 286 and 283.5 g/L was achieved for the unicellular alga Scenedesmus acuminatus grown in 7.5-L bench-scale and 1,000-L pilot-scale fermenters, respectively. The outdoor scale-up experiments indicated that heterotrophically grown S. acuminatus cells are more productive in terms of both biomass and lipid accumulation when they are inoculated in photobioreactors for lipid production as compared to the cells originally grown under photoautotrophic conditions. Technoeconomic analysis based on the pilot-scale data indicated that the cost of heterotrophic cultivation of microalgae for biomass production is comparable with that of the open-pond system and much lower than that of tubular PBR, if the biomass yield was higher than 200 g/L. This study demonstrated the economic viability of heterotrophic cultivation on large-scale microalgal inocula production, but ultrahigh-productivity fermentation is a prerequisite. Moreover, the advantages of the combined heterotrophic and photoautotrophic cultivation of microalgae for biofuels production were also verified in the pilot-scale.     

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。     

Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: A critical review

Microalgae have the ability to mitigate CO₂ emission and produce oil with a high productivity, thereby having the potential for applications in producing the third-generation of biofuels. The key technologies for producing microalgal biofuels include identification of preferable culture conditions for high oil productivity, development of effective and economical microalgae cultivation systems, as well as separation and harvesting of microalgal biomass and oil. This review presents recent advances in microalgal cultivation, photobioreactor design, and harvesting technologies with a focus on microalgal oil (mainly triglycerides) production. The effects of different microalgal metabolisms (i.e., phototrophic, heterotrophic, mixotrophic, and photoheterotrophic growth), cultivation systems (emphasizing the effect of light sources), and biomass harvesting methods (chemical/physical methods) on microalgal biomass and oil production are compared and critically discussed. This review aims to provide useful information to help future development of efficient and commercially viable technology for microalgae-based biodiesel production.     

Microalgae: a robust “green bio-bridge” between energy and environment

Microalgae are a potential candidate for biofuel production and environmental treatment because of their specific characteristics (e.g. fast growth, carbon neutral, and rich lipid accumulations). However, several primary bottlenecks still exist in current technologies, including low biomass conversion efficiency, bio-invasion from the external environment, limited or costly nutrient sources, and high energy and capital input for harvest, and stalling its industrial progression. Coupling biofuel production with environmental treatment renders microalgae a more feasible feedstock. This review focuses on microalgae biotechnologies for both bioenergy generation and environmental treatment (e.g. CO₂ sequestration and wastewater reclamation). Different intelligent technologies have been developed, especially during the last decade, to eliminate the bottlenecks, including mixotrophic/heterotrophic cultivation, immobilization, and co-cultivation. It has been realized that any single purpose for the cultivation of microalgae is not an economically feasible option. Combinations of applications in biorefineries are gradually reckoned to be necessary as it provides more economically feasible and environmentally sustainable operations. This presents microalgae as a special niche occupier linking the fields of energy and environmental sciences and technologies. The integrated application of microalgae is also proven by most of the life-cycle analysis studies. This study summarizes the latest development of primary microalgal biotechnologies in the two areas that will bring researchers a comprehensive view towards industrialization with an economic perspective.     

Microalgae for high-value compounds and biofuels production: A review with focus on cultivation under stress conditions

Microalgal biomass as feedstock for biofuel production is an attracting alternative to terrestrial plant utilization for biofuels production. However, today the microalgal cultivation systems for energy production purposes seem not yet to be economically feasible. Microalgae, though cultivated under stress conditions, such as nutrient starvation, high salinity, high temperature etc. accumulate considerable amounts (up to 60–65% of dry weight) of lipids or carbohydrates along with several secondary metabolites. Especially some of the latter are valuable compounds with an enormous range of industrial applications. The simultaneous production of lipids or carbohydrates for biofuel production and of secondary metabolites in a biorefinery concept might allow the microalgal production to be economically feasible. This paper aims to provide a review on the available literature about the cultivation of microalgae for the accumulation of high-value compounds along with lipids or carbohydrates focusing on stress cultivation conditions.     

Potential of Chlorella vulgaris culture for waste treatment from anaerobic biomass biodigestion at the Piaszczyna (Poland) integrated facility

Many strains of microalgae are potentially useful for industrial purposes. Microalgal biomass and microalgae‐derived substances are becoming valuable products with a widening range of applications including biofuels and human food. In this study, the possibility of using the methane waste from biomass biodigestion in the cultivation of Chlorella vulgaris biomass with simultaneous waste treatment was investigated. The methane waste from biomass biodigestion was obtained from a multifunctional facility (Piaszczyna, Poland) producing bioethanol from plant biomass with several steps to reuse the wastes, heat, and carbon dioxide. The growth and biomass yield, as well as photosynthetic performance of C. vulgaris on diluted waste, were similar to the results obtained on the standard mineral medium. The cultivation of C. vulgaris was the waste, treatment step that significantly reduced chemical oxygen demand. The results indicated that the waste contained micro‐ and macronutrients sufficient to sustain the growth of C. vulgaris cell culture up to 2 g of dry biomass per liter of culture. The results contributed to the development of the waste treatment step in the Piaszczyna facility that allowed for a further decrease in emissions and may lead to development of microalgae biomass‐based products in the facility portfolio.     

Isolation and Characterization of Taiwanese Heterotrophic Microalgae: Screening of Strains for Docosahexaenoic Acid (DHA) Production

Marine heterotrophic microalgal species which are potentially rich in docosahexaenoic acid (DHA, C22:6n−3) have been found in Taiwan; however, there was a lack of detailed analysis and characterization of these indigenous algae which is needed for the development of commercial applications. Hence, the objective of this study was to screen DHA-rich heterotrophic microalgae species indigenous to Taiwan for commercial purposes. Heterotrophic microalgae from a variety of marine habitats were isolated, cultivated, and then identified according to their 18S rRNA gene sequences and morphological characteristics. A comparison was made of their fatty acid profiles, fatty acid content, and amount of biomass. For the strain with highest DHA yield, the optimal growth conditions were determined in order to establish the best fermentation conditions for scale-up. In this study, 25 heterotrophic microalgal strains were successfully isolated from marine habitats around Taiwan. All of the isolated strains showed a close phylogenic relationship with the Thraustochytriaceae family according to their 18S rRNA gene sequences. GC/MS analysis discerned seven distinctive fatty acid profiles of these strains, with the production of eicosapentaenoic acid (C20:5n−3) ranging from 0.02 to 2.61 mg L⁻¹, and DHA ranging from 0.8 to 18.0 mg L⁻¹. An Aurantiochytrium strain BL10 with high DHA production was subsequently chosen for further manipulation. Under optimal growth conditions it could produce up to 59.0 g of dry biomass per liter of culture, with dry biomass containing 73% total fatty acid and 29% DHA, revealing BL10 as an excellent source of microbial DHA.     

Production of <Emphasis Type="Italic">Chlorella</Emphasis> biomass enriched by selenium and its use in animal nutrition: a review

Feedstuffs are routinely supplemented with various selenium sources, where organic forms of Se are more bio-available and less toxic than the inorganic forms (selenites, selenates). When the algae are exposed to environmental Se in the form of selenite, they are able as other microorganisms to incorporate the element to different levels, depending on the algae species. Technology of heterotrophic fed-batch cultivation of the microalga Chlorella enriched by organically bound Se was developed, where the cultivation proceeds in fermentors on aerated and mixed nutrient solution with urea as a nitrogen and glucose as a carbon and energy source. High volumetric productivity and high cell concentrations (about 70–100 g Chlorella dry mass l⁻¹) can be attained if nutrients and oxygen are adequately supplied. Addition of a small quantity of a new selenoprotein source-spray-dried Se-Chlorella biomass to the diet of farm animals had better effects on specific physiological and physical parameters of animals than selenite salt and was comparable with Se yeast added to the diet. This review introduces the importance of selenium for humans and animals, methods of Se determination, heterotrophic production of selenium-enriched Chlorella biomass in a fed-batch culture regime on organic carbon, and use of the biomass in animal nutrition.     

Season variability of iron effects on periodic culture of microalgae Dunaliella viridis Teod.

Effects of different iron concentrations (final concentrations of iron in Artari’s medium: 3.7, 37.0, 74.0, and 185.0 mmol·L⁻¹) on growth rate and contents of protein, triacylglycerides, and β-carotene in Dunaliella viridis cells at cultivation in different months were investigated. It was shown that the dose-dependent effects of iron were notable for season variability. In the 1^st experimental series (October, 2007), iron at researched concentrations did not affect growth rate of culture and protein, triacylglyceride, and β-carotene contents in cells. In experimental series conducted respectively in November 2007, December 2007, and February 2008, the dose-dependent stimulation of microalgae growth was observed. For each of these experimental series, there were particular dose dependences of protein, triacylglyceride, and β-carotene contents in microalgae cells at cultivation on media with iron at different concentrations. Meanwhile, for all of the four experimental series conducted in different months, variability of growth rate and analyzed parameters of microalgae Dunaliella viridis as control (cultivation without iron) was shown. It is suggested that these functional differences of control cultures of microalgae in different months caused variability in the dose-dependent effects of iron in a Dunaliella viridis culture. The possibility of iron usage for increasing microalgae biomass and for enriching it by β-carotene in Dunaliella viridis culture with initial low productivity and low β-carotene content is considered.     

Flue gas compounds and microalgae: (Bio-)chemical interactions leading to biotechnological opportunities

Flue gases are a resource yet to be fully utilised in microalgal biotechnology, not only to moderate the anthropogenic effects on our climate, but also to steer microalgal resource management towards innovative applications of microalgal biomass compounds. These gases, both untreated and treated into current discharge standards, contain CO₂, N₂, H₂O, O₂, NO_x, SO_x, C_xH_y, CO, particulate matter, halogen acids and heavy metals. To better steer and engineer flue gas-fed microalgal cultures, all these compounds need to be considered. Therefore, here, we review (i) the chemical composition and treatment technologies of flue gas, (ii) the uptake pathways and removal of the different compounds in microalgae reactors, and (iii) the tolerance and effects on microalgae of all flue gas compounds. By emphasising the interactions between microalgae and flue gas compounds, we envisage new pathways for microalgal biomass valorisation such as enzyme production for environmental technology, novel biogas production and biosequestration of minerals. Furthermore, we highlight fundamental and applied research niches that merit further investigation.     

Factors affecting lipid accumulation by <Emphasis Type="Italic">Nannochloropsis oculata</Emphasis> in a two-stage cultivation process

Reserve lipids of microalgae are promising for biodiesel production. However, optimization of cultivation conditions for both biomass yield and lipid production of microalgae is a contradictory problem because required conditions for both targets are different. In this study, a two-stage cultivation strategy is proposed to enhance lipid production of the microalga Nannochloropsis oculata. Biomass growth and lipid production were carried out in two separate and non-interacting stages. In first-stage cultivation, microalgae were cultivated in optimal conditions for cell growth. Then, microalgae were harvested and transferred into a growth-limited environment, thus enhancing lipid production of microalgae. Here, optimization of the lipid production stage (second stage) with respect to different levels of inoculum concentration, salinity of culture broth, and intensity of irradiance was performed. The results show that irradiance exhibits a significant influence on lipid production. The highest lipid productivity of 0.324 g L⁻¹ day⁻¹ was obtained with an inoculum concentration of 2.3 g L⁻¹, a salinity of 35 g L⁻¹, and an irradiance of 500 μmol photons m⁻² s⁻¹. The final yield of lipid obtained from the two-stage process was 2.82-times higher than that from traditional single-stage batch cultivation systems.     

以异养细胞作为种子的椭圆小球藻产油脂光自养培养优化

异养细胞种子/光自养培养方法是一种可异养培养的能源微藻培养的有效方法,但已有文献尚未从工艺优化角度考察其发展潜力。为了获得较高细胞密度的用于光自养培养的种子和提高光自养培养的细胞密度与油脂产率,对异养细胞种子/光自养培养的培养基和培养条件进行了优化。结果表明,采用优化后的培养基,椭圆小球藻在摇瓶中异养培养的最高藻细胞密度可达11.04 g/L,比在初始培养基条件下提高了28.0%,在5 L发酵罐中异养培养的藻细胞密度达到73.89 g/L;在2 L柱式光生物反应器中光自养培养的藻细胞密度、油脂含量和油脂产率分别达1.62 g/L、36.34%和6.1 mg/(L·h),油脂成分主要为含C16-C18碳链的脂肪酸,是制备生物柴油的理想原料。经过优化,异养细胞种子/光自养培养这一方法能够显著地提高椭圆小球藻产油脂的能力,这进一步表明异养细胞种子/光自养培养方法有望成为可异养的能源微藻的高效培养方式。     

Progress in physicochemical parameters of microalgae cultivation for biofuel production

Microalgae have been exploited for biofuel generation in the current era due to its enormous energy content, fast cellular growth rate, inexpensive culture approaches, accumulation of inorganic compounds, and CO₂ sequestration. Currently, research is ongoing towards the advancement of the microalgae cultivation parameters to enhance the biomass yield. The main objective of this study was to delineate the progress of physicochemical parameters for microalgae cultivation such as gaseous transfer, mixing, light demand, temperature, pH, nutrients and the culture period. This review demonstrates the latest research trends on mass transfer coefficient of different microalgae culturing reactors, gas velocity optimization, light intensity, retention time, and radiance effects on microalgae cellular growth, temperature impact on chlorophyll production, and nutrient dosage ratios for cellulosic metabolism to avoid nutrient deprivation. Besides that, cultivation approaches for microalgae associated with mathematical modeling for different parameters, mechanisms of microalgal growth rate and doubling time have been elaborately described. Along with that, this review also documents potential lipid-carbohydrate-protein enriched microalgae candidates for biofuel, biomass productivity, and different cultivation conditions including open-pond cultivation, closed-loop cultivation, and photobioreactors. Various photobioreactor types, the microalgae strain, productivity, advantages, and limitations were tabulated. In line with microalgae cultivation, this study also outlines in detail numerous biofuels from microalgae.     

Heterotrophic cultivation of microalgae for pigment production: A review

Pigments (mainly carotenoids) are important nutraceuticals known for their potent anti-oxidant activities and have been used extensively as high end health supplements. Microalgae are the most promising sources of natural carotenoids and are devoid of the toxic effects associated with synthetic derivatives. Compared to photoautotrophic cultivation, heterotrophic cultivation of microalgae in well-controlled bioreactors for pigments production has attracted much attention for commercial applications due to overcoming the difficulties associated with the supply of CO₂ and light, as well as avoiding the contamination problems and land requirements in open autotrophic culture systems. In this review, the heterotrophic metabolic potential of microalgae and their uses in pigment production are comprehensively described. Strategies to enhance pigment production under heterotrophic conditions are critically discussed and the challenges faced in heterotrophic pigment production with possible alternative solutions are presented.     

Production stability and biomass quality in microalgal cultivation – Contribution of community dynamics

The prospect of using constructed communities of microalgae in algal cultivation was confirmed in this study. Three different algal communities, constructed of diatoms (Diatom), green algae (Green), and cyanobacteria (Cyano), each mixed with a natural community of microalgae were cultivated in batch and semi‐continuous mode and fed CO₂ or cement flue gas (12–15% CO₂). Diatom had the highest growth rate but Green had the highest yield. Changes in the community composition occurred throughout the experiment. Green algae were the most competitive group, while filamentous cyanobacteria were outcompeted. Euglenoids, recruited from scarce species in the natural community became a large part of the biomass in semi‐steady state in all communities. High temporal and yield stability were demonstrated in all communities during semi‐steady state. Valuable products (lipids, proteins, and carbohydrates) comprised 61.5 ± 5% of ash‐free biomass and were similar for the three communities with lipids ranging 14–26% of dry mass (DM), proteins (15–28% DM) and carbohydrates (9–23% DM). Our results indicate that culture functions (stability, biomass quality) were maintained while dynamic changes occurred in community composition. We propose that a multispecies community approach can aid sustainability in microalgal cultivation, through complementary use of resources and higher culture stability.     

Statistical evaluation and modeling of cheap substrate-based cultivation medium of Chlorella vulgaris to enhance microalgae lipid as new potential feedstock for biolubricant

Chlorella vulgaris (C. vulgaris) microalga was investigated as a new potential feedstock for the production of biodegradable lubricant. In order to enhance microalgae lipid for biolubricant production, mixotrophic growth of C. vulgaris was optimized using statistical analysis of Plackett–Burman (P-B) and response surface methodology (RSM). A cheap substrate-based medium of molasses and corn steep liquor (CSL) was used instead of expensive mineral salts to reduce the total cost of microalgae production. The effects of molasses and CSL concentration (cheap substrates) and light intensity on the growth of microalgae and their lipid content were analyzed and modeled. Designed models by RSM showed good compatibility with a 95% confidence level when compared to the cultivation system. According to the models, optimal cultivation conditions were obtained with biomass productivity of 0.123 g L^?1 day^?1 and lipid dry weight of 0.64 g L^?1 as 35% of dry weight of C. vulgaris. The extracted microalgae lipid presented useful fatty acid for biolubricant production with viscosities of 42.00 cSt at 40°C and 8.500 cSt at 100°C, viscosity index of 185, flash point of 185°C, and pour point of ?6°C. These properties showed that microalgae lipid could be used as potential feedstock for biolubricant production.     

Effects of cassava starch hydrolysate on cell growth and lipid accumulation of the heterotrophic microalgae <Emphasis Type="Italic">Chlorella protothecoides</Emphasis>

Heterotrophic fermentation of microalgae has been shown to accumulate high amounts of microalgal lipids, which are regarded as one of the most promising feedstocks for sustainable biodiesel production. To increase the biomass and reduce the cost of microalgal culture, the purpose of this study was to evaluate the possibility of using cassava starch hydrolysate (CSH) instead of glucose as carbon source for heterotrophic culture of Chlorella protothecoides in flasks. First, the two-step enzymatic process of hydrolysis of cassava starch by α-amylase and glucoamylase was optimized; the conversion efficiency for cassava starch was up to 97.7%, and over 80% of CSH was glucose. Subsequently, we compared heterotrophic cultures of C. protothecoiedes using glucose or CSH as carbon source. The results demonstrated that when using CSH as the organic carbon source, the highest biomass and the maximum total lipid yield obtained were 15.8 and 4.19 g/L, representing increases of 42.3 and 27.7%, respectively, compared to using glucose as the organic carbon source. This suggests that CSH is a better carbon source than glucose for heterotrophic Chlorella protothecoides.     

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.     

Improving carbohydrate production of Chlorella sorokiniana NIES‐2168 through semi‐continuous process coupled with mixotrophic cultivation

Biofuels from microalgae is now a hot issue of great potential. However, achieving high starch productivity with photoautotrophic microalgae is still challenging. A feasible approach to enhance the growth and target product of microalgae is to conduct mixotrophic cultivation. The appropriate acetate addition combined with CO₂ supply as dual carbon sources (i.e., mixotrophic cultivation) could enhance the cell growth of some microalgae species, but the effect of acetate‐mediated mixotrophic culture mode on carbohydrate accumulation in microalgae remains unclear. Moreover, there is still lack of the information concerning how to increase the productivity of carbohydrates from microalgae under acetate‐amended mixotrophic cultivation and how to optimize the engineering strategies to achieve the goal. This study was undertaken to develop an optimal acetate‐contained mixotrophic cultivation system coupled with effective operation strategies to markedly improve the carbohydrate productivity of Chlorella sorokiniana NIES‐2168. The optimal carbohydrate productivity of 695 mg/L/d was obtained, which is the highest value ever reported. The monosaccharide in the accumulated carbohydrates is mainly glucose (i.e., 85–90%), which is very suitable for bio‐alcohols fermentation. Hence, by applying the optimal process developed in this study, C. sorokiniana NIES‐2168 has a high potential to serve as a feedstock for subsequent biofuels conversion.     

Growing Phototrophic Cells without Light

Many phototrophic microorganisms contain large quantities of high-value products such as n-3 polyunsaturated fatty acids and carotenoids but phototrophic growth is often slow due to light limitation. Some phototrophic microorganisms can also grow on cheap organic substrate heterotrophically. Heterotrophic cultivation can be well controlled and provides the possibility to achieve fast growth and high yield of valuable products on a large scale. Several strategies have been investigated for cultivation of phototrophic microorganisms without light. These include trophic conversion of obligate photoautotrophic microorganisms by genetic engineering, development of efficient cultivation systems and optimization of culture conditions. This paper reviews recent advances in heterotrophic cultivation of phototrophic cells with an emphasis on microalgae.