Comparison of biomass production and total lipid content of freshwater green microalgae cultivated under various culture conditions

The growth and total lipid content of four green microalgae (Chlorella sp., Chlorella vulgaris CCAP211/11B, Botryococcus braunii FC124 and Scenedesmus obliquus R8) were investigated under different culture conditions. Among the various carbon sources tested, glucose produced the largest biomass or microalgae grown heterotrophically. It was found that 1 % (w/v) glucose was actively utilized by Chlorella sp., C. vulgaris CCAP211/11B and B. braunii FC124, whereas S. obliquus R8 preferred 2 % (w/v) glucose. No significant difference in biomass production was noted between heterotrophic and mixotrophic (heterotrophic with light illumination/exposure) growth conditions, however, less production was observed for autotrophic cultivation. Total lipid content in cells increased by approximately two-fold under mixotrophic cultivation with respect to heterotrophic and autotrophic cultivation. In addition, light intensity had an impact on microalgal growth and total lipid content. The highest total lipid content was observed at 100 μmol m^?2s^?1 for Chlorella sp. (22.5 %) and S. obliquus R8 (23.7 %) and 80 μmol m^?2s^?1 for C. vulgaris CCAP211/11B (20.1 %) and B. braunii FC124 (34.9 %).     

Lipid and total fatty acid productivity in photoautotrophic fresh water microalgae: screening studies towards biodiesel production

Microalgae are considered as a promising feedstock for biomass production. The selection of the most suitable species is based on several key parameters such as lipid and fatty acid productivity. In the present study, the growth of different microalgae strains was examined in freshwater media for photoautotrophs suited for large-scale applications to identify the most suitable medium for each species. In the optimal medium, Scenedesmus obliquus showed the highest biomass productivity measured as increase of cell dry weight (0.25 g cellu dry weight (CDW) L^?1 day^?1), while Botryococcus braunii showed the highest lipid and total fatty acid content (430 and 270 mg g^?1 CDW, respectively) among the tested species. Regarding lipid and total fatty acid productivity, S. obliquus was the most lipid and total fatty acid productive strain with 41 and 18 mg L^?1 day^?1 during the exponential phase, respectively. Additionally, the proportion of saturated and monounsaturated fatty acids increased with duration of the incubation in S. obliquus, while polyunsaturated fatty acids decreased. These results nominate S. obliquus as a promising microalga in order to serve as a feedstock for renewable energy production.     

Improved Biomass and Hydrocarbon Productivity of Botryococcus braunii by Periodic Ultrasound Stimulation

Ultrasonic treatment was firstly found to accelerate both biomass and hydrocarbon productivities of Botryococcus braunii algal cells cultured in the shake flasks. The most effective sonication strategy was to subject the cells to three 5-min ultrasonic treatments at a 4-day interval using a fixed frequency of 40 kHz and power of 240 W, and the ultrasound-treated algal cells showed the highest biomass productivity of 0.043 g L^?1 day^?1 and the highest hydrocarbon productivity of 13.1 mg L^?1 day^?1 among all ultrasound treatments tested. The improved productivity was proved to be mainly due to the enhancement of both endogenous indole-3-acetic acid (IAA) biosynthesis and membrane permeability in the ultrasound-stimulated algal cells. The efficient ultrasonic stimulation strategy also showed good performance for the algal culture in a 2-L airlift bioreactor. Together, these results not only illustrate the immense potential for an enhanced understanding on ultrasound-stimulated algal cells but also as provide a powerful process intensification method to improve the biomass and hydrocarbon productivity of B. braunii in practice.     

An applicable nitrogen supply strategy for attached cultivation of Aucutodesmus obliquus

The “attached cultivation” method of microalgae in which the wet paste of algal biomass is attached onto supporting materials to form an immobilized biofilm layer, and the culture medium is supplied to this layer to provide nutrients and moisture for growth was highly efficient in biomass production and represents a promising technology to improve the biofuel industry. To optimize the nitrogen supply strategy for this attached cultivation method, the growth and total lipids accumulation properties for the green alga Aucutodesmus obliquus with this method were studied under different quantities of nitrogen source and different volumes of aqueous medium that continuously circulated inside the photobioreactor. Results showed that, compared with medium volume, the nitrogen quantity was a stronger factor affecting the growth and total lipid accumulation. An optimized nitrogen supply strategy for the attached cultivation of A. obliquus is proposed as circulating ca. 60 L of BG-11 medium containing 1/10 of nitrate concentration for 1 m² of cultivation surface. With this strategy, the attached A. obliquus accumulated biomass and total lipids simultaneously and obtained a high triacylglyceride productivity of 2.53 g m^?2 day^?1 in 7 days under subsaturated illumination of 100 μmol photons m^?2 s^?1. The water usage of 60 L m^?2 was potentially decreased to <2 L m^?2 if the nutrient supply was further improved. Dissolving the nitrogen source in small volume was the best way to efficiently utilize the nitrogen source with minimum of waste.     

Competition and succession between the oily alga Botryococcus braunii and two green algae Chlorella vulgaris and Chlamydomonas reinhardtii

Contamination by other algal species is a major problem in the culture of the oil-producing green alga Botryococcus braunii (Bb). In this study, the population interactions between Bb and two small green algae, Chlorella vulgaris (Cv) and Chlamydomonas reinhardtii (Cr), were separately investigated in two controlled experiments. Filtrates of Cv or Cr significantly stimulated the growth of Bb in the first 9–11 days when the filtrates were derived from a low- or medium-density algal culture. However, filtrates of Cv and Cr from the high-cell-density cultures significantly inhibited Bb growth from day 11 and 7, respectively. In Bb–Cv competition, the growth of Bb was significantly inhibited by Cv at the biomass ratio of Bb/Cv?=?1:1. However, when the Cv component reduced to 0.5 (Bb/Cv?=?1:0.5) or 0.25 (Bb/Cv?=?1:0.25), the growth of Bb was faster than that in the control in the first 13 days. Similarly, in Bb–Cr competition, the biomass ratios of 1:0.5 and 1:0.25 (Bb/Cr) favoured the growth of B. braunii in the first 15 days, but the growth of Bb decreased in the Bb/Cr ratio of 1:1 from day 15. By contrast, the growth of Cv and Cr were not affected by the presence of Bb in all biomass ratios. These results suggest that Cv and Cr could impact the growth of Bb by metabolites in the culture medium, and when the biomass of Bb is two to four times that of other algae, this oily alga becomes competitive over other algal species. This study reveals the mechanism of population dynamics of this oily alga competing with small and fast-growing algae. The results are applicable in reducing algal competition and regulating species succession between slow and fast-growing algae species.     

Productivity and biochemical composition of <Emphasis Type="Italic">Tetradesmus obliquus</Emphasis> and <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis>: effects of different cultivation approaches

The present work evaluated biomass productivity, carbon dioxide fixation rate, and biochemical composition of two microalgal species, Phaeodactylum tricornutum (Bacillariophyta) and Tetradesmus obliquus (Chlorophyta), cultivated indoors in high-technology photobioreactors (HT-PBR) and outdoors both in pilot ponds and low-technology photobioreactors in a greenhouse in southern Italy. Microalgae were grown in standard media, under nitrogen starvation, and in two liquid digestates obtained from anaerobic digestion of agro-zootechnical and vegetable biomass. P. tricornutum, cultivated in semi-continuous mode in indoor HT-PBRs with standard medium, showed a biomass productivity of 21.0?±?2.3 g m^?2 d^?1. Applying nitrogen starvation, the lipid productivity increased from 2.3 up to 4.5?±?0.5 g m^?2 d^?1, with a 24 % decrease of biomass productivity. For T. obliquus, a biomass productivity of 9.1?±?0.9 g m^?2 d^?1 in indoor HT-PBR was obtained using standard medium. Applying liquid digestates as fertilizers in open ponds, T. obliquus gave a biomass productivity (10.8?±?2.0 g m^?2 d^?1) not statistically different from complete medium such as P. tricornutum (6.5?±?2.2 g m^?2 d^?1). The biochemical data showed that the fatty acid composition of the microalgal biomass was affected by the different cultivation conditions for both microalgae. In conclusion, it was found that the microalgal productivity in standard medium was about doubled in HT-PBR compared to open ponds for P. tricornutum and was about 20 % higher for T. obliquus.     

Desiccation tolerance of Botryococcus braunii (Trebouxiophyceae,Chlorophyta) and extreme temperature tolerance of dehydrated cells

Botryococcus braunii Kützing, a green colonial microalga, occurs worldwide in both freshwater and brackish water environments. Despite considerable attention to B. braunii as a potential source of renewable fuel, many ecophysiological properties of this alga remain unknown. Here, we examined the desiccation and temperature tolerances of B. braunii using two newly isolated strains BOD-NG17 and BOD-GJ2. Both strains survived through 6- and 8-month desiccation treatments but not through a 12-month treatment. Interestingly, the desiccation-treated cells of B. braunii gained tolerance to extreme temperature shifts, i.e., high temperature (40 °C) and freezing (?20 °C). Both strains survived for at least 4 and 10 days at 40 and ?20 °C, respectively, while the untreated cells barely survived at these temperatures. These traits would enable long-distance dispersal of B. braunii cells and may account for the worldwide distribution of this algal species. Extracellular substances such as polysaccharides and hydrocarbons seem to confer the desiccation tolerance.     

Optimization of aeration for biodiesel production by <Emphasis Type="Italic">Scenedesmus obliquus</Emphasis> grown in municipal wastewater

Despite the significant breakthroughs in research on microalgae as a feedstock for biodiesel, its production cost is still much higher than that of fossil diesel. One possible solution to overcome this problem is to optimize algal growth and lipid production in wastewater. The present study examines the optimization of pretreatment of municipal wastewater and aeration conditions in order to enhance the lipid productivity of Scenedesmus obliquus. Results showed that no significant differences were recorded in lipid productivity of S. obliquus grown in primary settled or sterilized municipal wastewater; however, ultrasound pretreatment of wastewater significantly decreased the lipid production. Whereas, aeration rates of 0.2 vvm significantly increased lipid content by 51 %, with respect to the non-aerated culture, which resulted in maximum lipid productivity (32.5 mg L^?1 day^?1). Furthermore, aeration enrichment by 2 % CO₂ resulted in increase of lipid productivity by 46 % over the CO₂ non-enriched aerated culture. Fatty acid profile showed that optimized aeration significantly enhanced monounsaturated fatty acid production, composed mainly of C18:1, by 1.8 times over the non-aerated S. obliquus culture with insignificant changes in polyunsaturated fatty acid proportion; suggesting better biodiesel characteristics for the optimized culture.     

Growth and photosynthetic activity of <Emphasis Type="Italic">Botryococcus braunii</Emphasis> biofilms

Botryococcus braunii is a green microalga capable of producing large amounts of external long-chain hydrocarbons suitable as a source of biofuel. There have been several studies indicating that cultures of B. braunii can reduce the energy and water requirement for mass biofuel production, especially if non-destructive extraction methods for milking hydrocarbons are used. Growing microalgae as a raw material for biofuel using conventional liquid-based cultivation (i.e., raceway ponds) has yet to be shown to be economically successful. An alternative solid growth (biofilm) cultivation method can markedly reduce the energy requirements and costs associated with the harvesting and dewatering processes. We evaluated the growth of biofilms of several strains of B. braunii (from races A, B, L and S) and found that three of the four tested races successfully grew to stationary phase in 10 weeks with no contamination. Among all races, B. braunii BOT22 (race B) reached the highest biomass and lipid yields (3.80 mg dry weight cm^?2 day^?1 and 1.11 mg dry weight cm^?2). Irrespective of the race, almost all photosynthetic parameters (F _V /F ₀ , PI_ABS and the OJIP curve) showed that the biofilm cultures were more stressed during lag and stationary phases than in logarithmic phase. We also studied the Botryococcus biofilm profiles using confocal microscopy and found that this method is suitable for estimating the overall biomass yield when compared with gravimetric measurement. In conclusion, the growth characteristics (biomass and lipid) and photosynthetic performance of all races indicated that B. braunii BOT22 is the most promising strain for biofilm cultivation.     

Biomass, lipid content, and fatty acid composition of freshwater Chlamydomonas mexicana and Scenedesmus obliquus grown under salt stress

Two freshwater microalgae including Chlamydomonas mexicana and Scenedesmus obliquus were grown on Bold Basal Medium (BBM) with different levels of salinity up to 100 mM NaCl. The dry biomass and lipid content of microalgae were improved as the concentration of NaCl increased from 0 to 25 mM. Highest dry weight (0.8 and 0.65 g/L) and lipid content (37 and 34 %) of C. mexicana and S. obliquus, respectively, were obtained in BBM amended with 25 mM NaCl. The fatty acid composition of the investigated species was also improved by the increased NaCl concentration. At 50 mM, NaCl palmitic acid (35 %) and linoleic acid (41 %) were the dominant fatty acids in C. mexicana, while oleic acid (41 %) and α-linolenic acid (20 %) were the major fractions found in S. obliquus.     

Improvement of hydrocarbon recovery by spouting solvent into culture of Botryococcus braunii

Botryococcus braunii, a green microalga, is known to produce plentiful liquid hydrocarbons as promising biodiesel resources. However, the hydrocarbon extraction methods that have so far achieved have several problems such as low efficiency and high cost. In our study, a solvent-spouted extraction process integrated with photo-bioculture was designed for simultaneous realization of hydrocarbon extraction and cell culture in two phases. The n-octane was selected as the best solvent among several solvents because its biocompatibility was highest for B. braunii. As a result, high level of biomass and hydrocarbon, 4.17 and 893.79 mg/L, respectively, was attained at 100 mL/min of solvent recycling rate through three times of processes for 66 days. Moreover, formation of cell clump was suppressed in solvent extraction, cells were regenerated after it, and thus cell viability was maintained even after repeated cycles of it. Finally, this solvent-spouted culture process required the smaller cost due to reuse of the less solvent and regenerated cells, compared with the other conventional methods. Accordingly, this technique would be applicable to exploit the continuous extraction of hydrocarbon from the algal biomass, especially for application on a large scale.     

Effects of initial population density (IPD) on growth and lipid composition of Nannochloropsis sp.

The microalga Nannochloropsis sp. was cultured under different initial population densities (IPDs) ranging from 0.11 to 9.09 g L^?1. The IPD affected the biomass and lipid accumulation significantly. The algal cultured with higher IPD resulted higher biomass concentration (up to 13.07 g L^?1) in 10 days growth. The biomass productivity with 0.98 g L^?1 IPD was 0.75 g L^?1 d^?1 which was higher than that of other IPDs. For IPDs ranging from 0.11 to 0.98 g L^?1, with the increase of IPD, the biomass productivity increased, while for IPD over 0.98 g L^?1, the biomass productivity decreased. Lipid content of the algal culture started with 0.11 g L^?1 IPD reached to 42 % of dry weight. But with the increase of IPD, the lipid content decreased. Lipid composition was analyzed using thin layer chromatography coupled with flame ionization detection (TLC/FID). Seven lipid classes were identified and quantified. The main reserve lipid, triacylglyceride (TAG), accumulated under all different IPD conditions. However, with the increasing IPD values, TAG content decreased from 59.1 to 23.5 % of the total lipids. Based on these results, to obtain the maximal biomass productivity and lipid productivity of Nannochloropsis sp. in mass cultivation systems, it is necessary to select an appropriate IPD.     

Non-destructive hydrocarbon extraction from Botryococcus braunii BOT-22 (race B)

There is worldwide interest in developing algal biofuel. One main reason for the lack of success so far in producing a sustainable transport fuel from microalgae is the high cost of biomass processing, especially dewatering and oil extraction. There is also a significant cost involved in the energy content of the nutrient fertilisers required for biomass production. Non-destructive oil extraction or “milking” from algae biomass has the potential to bypass all of these hurdles. Using a “milking” strategy means that there would be no need for (a) biomass dewatering, (b) breaking cells for oil extraction and (c) addition of nutrients to the culture, resulting in a significant reduction in energy and fertiliser cost involved in production of biofuel from algae. We make use of the natural tendency of Botryococcus to produce external hydrocarbon in the extracellular matrix. In current study, we showed that external hydrocarbon from Botryococcus braunii BOT-22 can be non-destructively extracted using n-heptane (optimum contact time with n-heptane?=?20 min). We were able to recover almost the entire de novo-produced external hydrocarbons at 5- and 11-day intervals when the culture was maintained with or without 1 % CO₂ addition, respectively. This repeated non-destructive extraction of external hydrocarbon of B. braunii was possible for up to 70 days when 1 % CO₂ was supplied to the culture. When CO₂ was limited, a 70 % lower external hydrocarbon productivity was achieved using the same process. Although the productivity of external hydrocarbon of 9.33 mg L^?1 day^?1 of the “milked” culture is low in these un-optimised cultures, it was 1.3?±?0.2-fold higher compared with that of a conventional semicontinuous culture, showing the potential of this method.     

Low temperature and <Emphasis Type="Italic">Daphnia</Emphasis>-associated infochemicals promote colony formation of <Emphasis Type="Italic">Scenedesmus obliquus</Emphasis> and its harvesting

Objective

To explore the combined effects of temperature and Daphnia-associated infochemicals on colony formation of Scenedesmus obliquus to faciliate harvesting the algal biomass.

Results

A three-parameter modified Gaussian model fitted the changes of the number of cells per particle in S. obliquus induced by Daphnia culture filtrate well under any temperature. Decreases in temperature enhanced the induced–colony formation of Scenedesmus. The maximum colony size at 15–25 °C was significantly larger than those at 30–35 °C. An additional 1 or 2 days at low temperature was needed to reach the maximum colony size, which indicates the best harvest time for algal biomass.

Conclusion

Induced-colony formation of Scenedesmus by Daphnia culture filtrate at 15–25 °C is recommended to settle algal cells. This condition facilitates harvesting the biomass.

     

Interspecific biodiversity enhances biomass and lipid productivity of microalgae as biofuel feedstock

Large improvements in biomass and lipid production are required to make massive scale algal biodiesel production an economic reality. The application of the biodiversity strategy to enhance algal biomass as biofuel feedstock is little. The algal diversity was manipulated in this study to investigate the effects of a combination of biodiversity complementarity and a new medium consisting of seawater and agricultural fertilizer on lipid productivity. The algae diverse community includes two strains of Dunaliella salina (Dunaliella salina 19/30 and 19/18) and three species of Nannochloropsis (Nannochloropsis oculata, Nannochloropsis salina, and Nannochloropsis gaditana). The results showed that the most diverse community (5 species) produced an average of sixfold more biomass in the new medium than did the standard f/2 culture medium. The most diverse polyculture had the highest growth rate (1.01 day^?1), biomass (1.2 g L^?1), and lipid productivity (0.31 g L^?1 day^?1). The assessment of algal polycultures relative to monocultures is particularly interesting and novel for this biofuel field, and the observations that these polycultures resulted in significant lipid productivity improvements are very useful addition to the biofuel research. The possible mechanism (resource diversity) to explain the synergy in mixed cultures warrants further investigation.     

重金属铅与两种淡水藻的相互作用

为了研究重金属铅与淡水藻类之间的相互作用,采用不同Pb2+浓度处理铜绿微囊藻(Microcysis aeruginosa Kutz.)和斜生栅藻[Scenedesmus obliquus(Turp.)Kutz.],分别对两种藻的生物量、藻液电导率、O-·2含量、过氧化物酶(POD)、过氧化氢酶(CAT)活性以及藻对Pb2+的吸收作用等进行了测定,并通过扫描电镜观察了不同Pb2+浓度处理下两种藻细胞的表面结构。结果显示:(1)Pb2+浓度低于3 mg/L促进铜绿微囊藻生长,高于9 mg/L抑制其生长;但在3—12 mg/L范围内,Pb2+均明显抑制了斜生栅藻的生长,说明斜生栅藻对Pb2+毒性的敏感程度要高于铜绿微囊藻。(2)受到铅离子的胁迫,两种藻细胞膜通透性均有一定改变,扫描电子显微镜的照片观察,两种藻细胞表面的絮状物随着Pb2+的升高而增多,尤其是斜生栅藻细胞结构改变明显,多数细胞变形破裂;同时,O-·2含量升高,POD、CAT活性早期均可随Pb2+的增加而上升,表明氧自由基的产生增多以及由其引起的细胞生理生化改变可能是铅离子作用于藻细胞的主要机制。(3)两种淡水藻对Pb2+均有吸收作用,单位量藻细胞内,斜生栅藻对Pb2+的吸收能力好于铜绿微囊藻。所有结果提示:斜生栅藻不仅可以作为对重金属敏感的指示生物来监测水体Pb2+污染程度;同时由于斜生栅藻比铜绿微囊藻具有更好的Pb2+吸收能力,因此还可以利用斜生栅藻作为处理水体Pb2+的生物材料。     

Improved algal oil production from Botryococcus braunii by feeding nitrate and phosphate in an airlift bioreactor

To improve biomass and microalgal oil production of Botryococcus braunii, fed‐batch culture was investigated in an airlift photobioreactor. The optimal feeding time of the fed‐batch culture was after 15 days of cultivation, where 1.82 g/L of the microalgal biomass was obtained in the batch culture. Nitrate nutrient was the restrictive factor for the fed‐batch cultivation while phosphate nutrient with high concentration did not affect the microalgal growth. The optimal mole ratio of nitrate to phosphate was 34.7:1, where nitrate concentration reached the initial level and phosphate concentration was one quarter of its initial level. With one feeding, the biomass of B. braunii reached 2.56 g/L after 18 days. Two feedings in 2‐day interval enhanced the biomass production up to 2.87 g/L after 19 days of cultivation. The hydrocarbon content in dry biomass of B. braunii kept at high level of 64.3% w/w. Compared with the batch culture, biomass production and hydrocarbon productivity of B. braunii were greatly improved by the strategic fed‐batch cultivation.     

Effect of different media on exopolysaccharide and biomass production by the green microalga Botryococcus braunii

Botryococcus braunii is a colonial green microalga with recognized potential to synthesize lipids and hydrocarbons for biofuel production. Besides this ability, this microalga also produces exopolysaccharides (EPS). Nevertheless, there are few reports about their biotechnological aspects and industrial applications. In this study, the effect of the nutritional conditions was examined by using two different culture media (BG11 and D medium). To our knowledge, the latter has not been reported before for culturing B. braunii. After 49 days of incubation, the final production of EPS was found to be statistically higher (P < 0.05) in the D medium (0.549?±?0.044 g L^?1) than in BG11 (0.336?±?0.009 g L^?1). On the contrary, the biomass production was found to be higher in BG11 (1.019?±?0.051 g L^?1) than in the D medium (0.953?±?0.056 g L^?1). However, this difference was not statistically significant. The difference in salinity and nitrogen concentration between both media is suggested as the main factor involved in the EPS and biomass results. FTIR spectra of B. braunii EPS from both media revealed presence of uronic acids and absence of amino and sulfate groups. Despite the similarity between both spectra, there were some different signals (at 1,921.52 and 720.60 cm^?1) which may mean a difference in glycosyl composition.     

Phycoremediation of dairy and winery wastewater using <Emphasis Type="Italic">Diplosphaera</Emphasis> sp. MM1

A new green microalgal species was isolated, identified and investigated for its biomass production and nutrient removal efficiency in dairy and winery wastewater in this study. The 18S rRNA-based phylogenetic analysis revealed that this new strain is a Diplosphaera sp. and was designated strain MM1. The growth of this strain was evaluated in different diluted dairy and winery wastewaters. The highest algal biomass production (up to 2.3 g L^?1) was obtained in dairy wastewater (D3; dairy wastewater 1:2 deionised water) after 14 days of culture. However, for winery wastewater, the highest algal biomass production (up to 1.46 g L^?1) was obtained in wastewater combination W2 (winery wastewater 1:1 deionised water) after 14 days of culture. Turbid dairy wastewater with high concentration of nitrogen and phosphorous slowed down the initial growth of the alga. However, at the end of day 14, biomass production was nearly twofold higher than that of winery wastewater. The findings from both types of wastewater suggest that Diplosphaera sp. MM1 has potential for its application in generating biomass with simultaneous remediation of nutrient-rich wastewater.