A new lipid-rich microalga Scenedesmussp. strain R-16 isolated using Nile red staining: effects of carbon and nitrogen sources and initial pH on the biomass and lipid production
© Ren et al.; licensee BioMed Central Ltd. 2013
Received: 24 June 2013
Accepted: 2 October 2013
Published: 6 October 2013
Biodiesel production from oleaginous microalgae shows great potential as a promising alternative to conventional fossil fuels. Currently, most research focus on algal biomass production with autotrophic cultivation, but this cultivation strategy induces low biomass concentration and it is difficult to be used in large-scale algal biomass production. By contrast, heterotrophic algae allows higher growth rate and can accumulate higher lipid. However, the fast-growing and lipid-rich microalgae that can be cultivated in heterotrophic system for the industrial application of biodiesel production are still few. Traditional solvent extraction and gravimetric determination to detect the microalgal total lipid content is time-consuming and laborious, which has become a major limiting factor for selecting large number of algae specimens. Thus, it is critical to develop a rapid and efficient procedure for the screening of lipid-rich microalgae.
A novel green microalga Scenedesmus sp. strain R-16 with high total lipid content was selected using the Nile red staining from eighty-eight isolates. Various carbon sources (fructose, glucose and acetate) and nitrogen sources (nitrate, urea, peptone and yeast extract) can be utilized for microalgal growth and lipid production, and the optimal carbon and nitrogen sources were glucose (10 g L-1) and nitrate (0.6 g L-1), respectively. Compared to autotrophic situation, the strain R-16 can grow well heterotrophically without light and the accumulated total lipid content and biomass reached 43.4% and 3.46 g L-1, respectively. In addition, nitrogen deficiency led to an accumulation of lipid and the total lipid content was as high as 52.6%, and it was worth noting that strain R-16 exhibited strong tolerance to high glucose (up to 100 g L-1) and a wide range of pH (4.0-11.0).
The newly developed ultrasonic-assisted Nile red method proved to be an efficient isolation procedure and was successfully used in the selection of oleaginous microalgae. The isolated novel green microalgal strain R-16 was rich in lipid and can live in varied and contrasting conditions. The algae appeared to have great potential for application in microalgae-based biodiesel production.
KeywordsBiodiesel Heterotrophy Isolation Lipid Microalga Nile red
Conventional fossil fuels, such as petroleum, coal and natural gas, still play a dominant role in the global energy consumption . However, it is well known that these traditional fuels are non-renewable with depleting reserves and increasing cost [2, 3]. The most promising method to meet the growing demand for energy is to explore economically feasible and alternative fuels [4, 5]. Recently, biodiesel exhibits great potential and attracts extensive interest as it is carbon-neutral and environment friendly . Traditional feedstock of biodiesel contains plant oils (canola, corn, soybean, oil palm, coconut, etc.) and animal fats . Nevertheless, such raw materials may compete with food supply, increase the utilization of limited farmland, and require long time to harvest which is hard to satisfy the large and long-term global energy demand .
Nowadays, biodiesel production technology from microalgae is widely considered as a potential and efficient method since a number of advantages, such as the simple cellular structure of microalgae, short production cycle, high intracellular lipid content, and fast growth rate [8, 9]. In addition, microalgae can be cultivated on non-arable land which could reduce the demand for farm-land and avoid the competition with food/feed crops . Though many microalgal strains have been isolated and established to be rich in neutral lipid, there are still some unknown species or strains present in various local environments with the potential of applying in the production of biodiesel.
Microalgal biomass production has largely been obtained by autotrophic cultivation in open pond or closed photo-bioreactor under natural or artificial source of light [11, 12]. Nevertheless, the cell density of this culture strategy is low and the light requirement is high, and these bottlenecks make it hard to be applied in large-scale algal biomass production . Compared to photoautotrophy, heterotrophic cultivation allows higher algal growth rate and enables microalgae to accumulate higher biomass and amounts of lipid using less time in the absence of light, which is critical for reducing the microalgal biomass production cost . However, only a few microalgae species adapt to heterotrophic cultivation, and most of them belong to the genus Chlorella. As such, it is important to screen more fast-growing and lipid-rich microalgae that can be cultivated in heterotrophic system for the industrial application of biodiesel production.
On the other hand, current methods to determine the total lipid content for selecting large number of algae specimens are complicated and generally contain extraction, purification, concentration and quantification of lipids . This process is laborious and the lipid components decompose easily . Recently, rapid and efficient screening methods are essential to decide novel and proper algae candidates. Nile red is a lipophilic fluorescent dye and has been used in algal biodiesel production to detect and quantify the lipid content of many microalgal strains . Furthermore, some researchers further modify the Nile red fluorescence technique to improve the lipid staining efficiency and obtain satisfactory outcomes [15, 16]. However, up to now, the information about using the modified Nile red method for screening of lipid-rich microalgal species or strains is still limited.
So, this study developed a novel ultrasonic-assisted staining procedure for algal sample analysis. The isolated microalgal strain was further investigated in heterotrophic cultures for lipid production with high efficiency. Moreover, the main factors (carbon source, nitrogen source and initial pH) were systematically studied and optimized for high biomass and lipid production. In addition, this work also compared the lipid and biomass production ability of the selected strain with those previously reported.
Results and discussion
Algal isolation with improved Nile red method
Identification of the microalga
To further determine the taxonomic position, molecular phylogenetic analysis was used to confirm the isolated strain. The 18S rRNA gene complete sequence of strain R-16 consisting of 1,419 bases was determined and submitted to the GenBank (Accession No.: KC859922). In the phylogram (Additional file 1: Figure S1), the 18S rDNA sequence of strain R-16 confirmed its identification as Scenedesmus sp.. The 18S rRNA gene complete sequence of strain R-16 exhibited 100% similarity to that of Scenedesmus abundans strain UTEX 343 (Accession No.: X73995.1), and the nucleotides of strain UTEX 343 (1,794 bases) covered all nucleotides of strain R-16 (1,419 bases). However, no identification could be made based on the ITS sequence comparison because the lack of molecular data for the Scenedesmus abundans in Genbank. The ITS sequence of strain R-16 shared 99% sequence similarity with that of Desmodesmus sp. Tow10/11 T-2 W (Accession No.: DQ417553.1), an uncharacterized Desmodesmus specie (Additional file 2: Table S1). Thus, more specific phylogenetic characterization cannot be given, and the microalga was called Scenedesmus sp. strain R-16 and used in the following experiments.
Effects of carbon and nitrogen sources
In addition, strain R-16 can utilize both inorganic and organic nitrogen sources for growth and lipid accumulation (Figure 3b and Additional file 3: Figure S2b). Sodium nitrate led to the highest biomass concentration of 3.47 g L-1 and specific growth rate of 0.821 d-1, followed by that achieved with the medium supplemented with urea (2.98 g L-1/0.781 d-1) and yeast extract (2.38 g L-1/0.727 d-1). The maximum total lipid content of 44.8% occurred when peptone was utilized as the nitrogen source, but the biomass concentration and specific growth rate were only 1.16 g L-1 and 0.552 d-1, respectively. It might be due to that inefficient utilization of peptone could result in the N-starvation of algal cells which induced higher total lipid content of algae . Above results showed that glucose and sodium nitrate were the best carbon and nitrogen source for both growth and lipid production of strain R-16 under the investigated conditions.
Effect of glucose
Effects of sodium nitrate and initial pH
The pH was another important factor which had great effect on the properties of microbial surface and flocculation, and such influence can change biochemical metabolism and the form of enzyme system . In the present research the initial pH varied from 3.0 to 12.0 (Figure 5b). This experiment found an interesting phenomenon, that is, the strain R-16 had strong pH tolerance and can grow well in a wide range of pH (4.0-11.0). Furthermore, the total lipid content of algal cells was similar from pH 6.0 to pH 11.0, while the total lipid content decreased at pH 4.0. At two extreme pH (3.0 and 12.0), the algal cells showed poor growth and lipid productivity. These results indicated that the algal growth and lipid accumulation were slightly affected by the pH of the medium (pH 6.0-11.0), which showed the potential of using strain R-16 for the treatment of wastewater or waste biomass into lipid in a wide pH range. In addition, similar phenomenon was also observed by a few researchers with various algal strains (Chlorella sorokiniana and Asterarcys quadricellulare) in autotrophic situation [20, 25]. However, in this study the strain R-16 can grow well in heterotrophic culture condition and also exhibited amazing pH tolerance, which might be caused by self-metabolism regulation or special structure of the microalgae. Nevertheless, up to now, the mechanism of this process has not yet been given, and the detailed explanation needed to be further investigated in the future work.
Growth and lipid production under various culture modes
Comparison of lipid production capability with relevant studies
Comparison of biomass and lipid production with glucose as carbon source
Glucose concentration (g L-1)
Biomass concentration (g L-1)
Total lipid content (%DW)
Lipid productivity (mg L-1d-1)
Chlorella sorokiniana Shih.et Krauss MIC-G5
Chlorella vulgaris C4-3
Chlorella saccharophila UTEX 247
Chlorella sorokiniana CCTCCM209220
Monoraphidium sp. FXY-10
In addition, a critical challenge of heterotrophic microalgae derived from the high cost of culture medium, especially the carbon sources . Most researchers reduced the cost of algal cultivation by using some cheap and easily available feedstock (such as starch, wastewater and cellulose-hydrolyzed solution) and gained promising results [29, 30]. It was found that some microalgae like strain R-16 can utilize the volatile fatty acids (VFAs) as the substrate for growth and lipid accumulation . In the production of other bio-energy (such as bio-hydrogen, bio-methane, bio-ethanol, bio-butanol and bio-electricity), there are many VFAs left in the effluent [31–33]. Take bio-hydrogen production for example, the degradation of large molecular substrates by dark-fermentative bacteria can generate many VFAs, which mainly contain acetic acid, propionic acid and butyric acid . However, VFAs in effluent may decrease the pH of reaction system and induce inhibition effect to the microorganisms or pollution to the environment. Photo-fermentative bacteria can utilize the VFAs from dark-fermentation as electron donors and produce hydrogen in the presence of light . Currently, most studies focused on the combination of dark- and photo-fermentation to alleviate the end-product inhibition and improve the substrate conversion efficiency [34–36]. Though the combined system can markedly increase the theoretical hydrogen yield to 12 mol mol-1 glucose-1, the experimental energy conversion efficiency of the combined system is still not high enough for the practical reality of biological hydrogen production . Furthermore, it should be noted that the photo-fermentative bacteria need additional light with low photosynthetic efficiency (less than 10%), which could greatly increase the operating cost . By contrast, above mentioned VFAs in effluent can be further utilized by microalgae without light for lipid production and energy recovery, whereas so far related research on this field was lacking. So, it was a promising strategy to combine the microalgal lipid production with one or more other bio-energy production methods to reduce the cost of algal cultivation and increase the energy recovery of the whole process.
The newly developed ultrasonic-assisted Nile red method was applied in the screening of lipid-rich microalgae and proved to be an efficient isolation procedure. Based on this method, a novel green microalgal strain, Scenedesmus sp. R-16, was isolated and characterized from 88 microalgae isolates. Strain R-16 can be acclimatized to high glucose (up to 100 g L-1) and a wide range of pH (4.0-11.0). Many carbon and nitrogen sources can be used by strain R-16 for growth and lipid production. By using the optimum carbon source (10 g L-1 of glucose) and nitrogen source (0.6 g L-1 of sodium nitrate), strain R-16 exhibited high biomass of 3.46 g L-1and total lipid content of 43.4% under heterotrophic condition. When nitrogen was scarce, total lipid content could reach a maximum value of 52.6%. Compared with other reported oleaginous algal strains, Scenedesmus sp. R-16 showed great potential in the lipid production for renewable biodiesel production.
Isolation and purification of the microalga
Samples of soil were collected from Harbin, Heilongjiang province, China, and then were inoculated in autoclaved BG11 medium at 25 ± 1°C under cool white fluorescent light until algal growth was detected . The pre-cultured samples were diluted and streaked on BG11 medium-enriched agar plates. Individual colonies were picked up and cultured in liquid BG11 medium that contained glucose using the same culture conditions described above. The streaking and inoculation procedures were repeated about 3–4 times until pure cultures were attained.
Genomic DNA was extracted with a SK1375 kit according to the manufacturer’s instructions . The 18S rDNA and ITS regions were amplified using the primers described in Additional file 4: Table S2 [41, 42]. The 25 μl PCR reaction system contained approximately 1 μl of template DNA, 0.5 μl (10 μmol L-1) of forward primer, 0.5 μl (10 μmol L-1) of reverse primer, 0.5 μl dNTP mixer (10 mmol L-1 each), 1.0 U of Ex Taq DNA polymerase and 2.5 μl 10 × Ex Taq PCR buffer. Amplification conditions were performed as follows: 5 min at 94°C, followed by 35 cycles of denaturation at 94°C for 30 s, 35 s annealing at 55°C, and 1 min extension step at 72°C with a final extension of 8 min at 72°C. The PCR products were separated by electrophoresis on 1.0% agarose gel. Afterwards, bands were extracted from gel and purified using a PCR purification kit (SK1131 kit). The purified PCR products were ligated into the pUCm-T followed by transforming into Escherichia coli competent cells (SK2301 kit). Recombinant plasmid was extracted from transformed E. coli with a SK1191 kit following the guidelines of the manufacturer . All kits were provided by Sangon Biotech (Shanghai) Co., Ltd., (Shanghai, China). Sequence alignment and analysis of the similarity of the18S rRNA and ITS gene were performed with BLAST in GenBank database.
In the autotrophic culture, algal cells in the stationary phase were inoculated into 250 ml Erlenmeyer flasks containing 150 ml BG11 medium, which had been adjusted to near neutral pH and autoclaved at 121°C for 15 min. The microalgae were cultured at 25 ± 1°C using incubator shaker at 130 rpm with serial florescence light of around 5000 lux. For heterotrophic culture, seven carbon sources (fructose, maltose, glucose, acetate, propionate, butyrate, sucrose) were separately supplemented to the liquid culture medium. The initial concentrations of all carbon sources were calculated as the same carbon atom number of 10 g L-1 glucose. Beef extract, urea, peptone, ammonium chloride, sodium nitrate, L-cysteine, yeast extract and sodium glutamate were chosen as the nitrogen sources with the initial concentrations computed as the same nitrogen atom number of 0.6 g L-1 sodium nitrate. The glucose concentration, sodium nitrate concentration and initial pH were in the range of 5–100 g L-1, 0.2-1.0 g L-1 and 3.0-12.0, respectively. Each culture was cultured using the same conditions described in the autotrophic culture without light exposure. The initial algal concentration of both autotrophic and heterotrophic trials was approximately 0.1 g L-1. After reaching the stationary phase (defined by no further increase in cell concentration), the cultures were harvested and analyzed for biomass and lipid production. All experiments were conducted in triplicate, and results were expressed as means of the replicates along with standard deviation (± SD).
Nile red staining
Eighty-eight algal strains that had high biomass were chosen for the determination of lipid content. Based on preliminary procedure for improved Nile red staining (data not published), the selected cells (5 ml) were centrifuged at 8,000 g for 10 min and washed with distilled water several times. Then the collected cells were re-suspended in a 10 ml tubes and pretreated with an ultrasonic processor (Sonics VCX130PB, USA). Furthermore, 15 μl of Nile red solution (0.5 mg ml-1 in acetone) was added to 5 ml of algal suspensions and gently vortexed for 1 min. After 15 min of incubation in darkness, the fluorescence of the suspension was measured with a fluorescence spectrophotometer (JASCO FP-6500, Japan). According to preliminary experiments, the excitation and emission wavelengths for the fluorescence determination were selected as 530 and 568 nm, respectively. Unstained cells and Nile red alone were used as the autofluorescence control. The relative fluorescence intensity of Nile red was attained after subtraction of both the autofluorescence of microalgae and the self-fluorescence of Nile red.
Algal growth kinetics
where dX/dt was the microalgal growth rate; k c was the maximum specific growth rate of the microalgae, X was the biomass concentration of microalgae, X max was the maximum cell concentration.
where N 2 and N 1 represented the DW values at the time t 2 and t 1 , respectively.
Scanning electron microscopy
- 18S rRNA:
18S ribosomal ribonucleic acid
- 18S rDNA:
18S ribosomal deoxyribonucleic acid
Internal transcribed spacer
Conversion ratio of glucose to oil
Volatile fatty acids
Polymerase chain reaction
Basic local alignment search tool
This study is supported by the national Natural Science Foundation of China (No. 51106040), project 51121062 (National Creative Research Groups), and Academician Workstation Construction in Guangdong Province (No. 2012B090500018).
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