Bioprocessing of Stichococcus bacillaris strain siva2011
© Sivakumar et al.; licensee BioMed Central Ltd. 2014
Received: 26 July 2013
Accepted: 17 March 2014
Published: 15 April 2014
Globally, the development of a cost-effective long-term renewable energy infrastructure is one of the most challenging problems faced by society today. Microalgae are rich in potential biofuel substrates such as lipids, including triacylglycerols (TAGs). Some of these algae also biosynthesize small molecule hydrocarbons. These hydrocarbons can often be used as liquid fuels, often with more versatility and by a more direct approach than some TAGs. However, the appropriate TAGs, accumulated from microalgae biomass, can be used as substrates for different kinds of renewable liquid fuels such as biodiesel and jet fuel.
This article describes the isolation and identification of a lipid-rich, hydrocarbon-producing alga, Stichococcus bacillaris strain siva2011, together with its bioprocessing, hydrocarbon and fatty acid methyl ester (FAME) profiles. The S. bacillaris strain siva2011 was scaled-up in an 8 L bioreactor with 0.2% CO2. The C16:0, C16:3, C18:1, C18:2 and C18:3 were 112.2, 9.4, 51.3, 74.1 and 69.2 mg/g dry weight (DW), respectively. This new strain produced a significant amount of biomass of 3.79 g/L DW on day 6 in the 8 L bioreactor and also produced three hydrocarbons.
A new oil-rich microalga S. bacillaris strain siva2011 was discovered and its biomass has been scaled-up in a newly designed balloon-type bioreactor. The TAGs and hydrocarbons produced by this organism could be used as substrates for jet fuel or biodiesel.
KeywordsAlgae Bioreactor Hydrocarbon Jet fuel Triacylglycerol
Worldwide consumption of crude oil is predicted to grow continuously. It is clear that in spite of improvements in the recovery of traditional fossil fuels, alternative renewable energy resources will at some point be needed. Moreover, such renewable fuels offer the prospect of minimizing increases in atmospheric CO2 by recycling carbon from the atmosphere back into a viable liquid fuel (or perhaps eventually sequestering it entirely). Over a large number of cycles, the net effect could be a significant reduction in the addition of CO2 into the atmosphere compared to continued reliance only on fossil fuels. A wide variety of existing biofuel technologies have been tested, but none have proven to provide a suitable source of replacement liquid fuels. Although current alternatives such as ethanol and biodiesel can provide carbon neutrality, fuels derived largely from normally edible plant sources affect the food supply negatively [1–3]. For these reasons algae feedstocks are being explored as an alternative . The development of a suitable algal-based jet fuel from algal biomass may also impact air transportation.
The jet fuel approach is to chemically process triacylglycerols (TAGs) to alkanes. This could be done by catalytic hydrotreating, breaking the TAG molecule and removing the oxygen to form alkanes. While this product meets diesel specifications, it can be further upgraded into jet fuel or naphtha by hydrocracking, isomerization and catalytic reforming . The by-product propane can be used for residential central heating. However, not all microalgae are capable of producing sufficient TAGs and hydrocarbons for effective fuel production. While others might produce abundant TAGs, they might not necessarily be the optimum TAGs for production of high-value products such as aviation fuel. For production of such specialized fuels, the selection of the algal species is the key to success. Carbon profiles for selecting algal strains  and catalytic hydrothermal decarboxylation of fatty acids for aviation fuel  have been studied.
Recently, Stichococcus bacillaris Naegeli was proposed as a potential and dedicated candidate for use in fuel production . S. bacillaris is a green soil microalga which includes over 14 species . Cells are approximately 2 to 3 μm in diameter. The state of filamentous or unicellular structures depends on salinity . This species can grow both in freshwater and seawater with different growth kinetics , while tolerating high salinities . In addition, this alga has adapted to low temperatures and is found in ice-free areas of Antarctica . Moreover, it also contains high NADPH-GDH activity , low CO2 resistance  and has unique microtubule organization in prophase . The NADPH-GDH plays an important role in photosynthetic microalgae, which is associated with photoregulation and the incorporation of ammonia into amino acids. The changes in NADPH-GDH were shown in different culture conditions such as photoautotrophic, heterotrophic and mixotrophic . Compared to ammonium, nitrate-grown S. bacillaris had higher activity of NADPH-GDH . S. bacillaris is fairly abundant globally, can remove heavy metals from hazardous environments  and is also capable of biotransforming phenols . These characteristics suggest that S. bacillaris could minimize water contamination or improve water quality.
In addition, this organism has a short life cycle and is tolerant to different ranges of pH. Most importantly, over 30% of its dry mass can be produced as oil that can be readily converted to biodiesel . Moreover, this alga produced a high percentage of C16 to C18 carbon fatty acids. Therefore, the goal was to isolate Stichococcus species for the study of aviation fuel. Other proposed algal strains either produced triterpene hydrocarbons that are difficult to convert cost-effectively to usable fuels or grew too slowly to be useful [22, 23]. Some other TAGs are produced from algae but they typically yield a low biomass . Thus, the aim of this research has been: 1) to isolate new Stichococcus algal species producing significant quantities of lipids and hydrocarbons, especially those suitable for production of aviation fuel; and 2) to evaluate the scale-up potential of this alga in a new design balloon-type bioreactor.
Results and discussions
Stichococcus bacillarisstrain siva2011 identification
Bioreactor culture of S. bacillarisstrain siva2011
On day 6 with 0.2% CO2, a maximum biomass of 3.79 g/L dry weight (DW) was achieved in 8 L and 3.45 g/L DW in 4 L, respectively. Since the new strain requires a very low 0.2% of CO2, the input cost on large-scale could be minimized. When compared to 0.5% CO2, algal cells grown in a 4 L bioreactor were higher in biomass, with 0.55, 0.986 and 1.45 g/L DW in the 0.05, 0.1 and 0.2% CO2, respectively. Similarly, in the 8 L bioreactor, biomass accumulation was 0.793, 1.107 and 1.79 g/L DW after 6 days of culture. Both growth and pH kinetic trends were similar in 4 L and 8 L bioreactors. The data supports the notion that this strain does not require light intensity over 15 to 30 μE m-2 s-1; therefore, achieving high density biomass may not be a problem. However, the optimization of air flow is needed for better media circulation and growth. The media circulation is critical for efficient photosynthesis. The oil-rich alga Ettlia oleoabundans accumulates 2.28 g/L dry biomass in BBM in approximately 22 to 27 days , while S. bacillaris strain siva2011 accumulates approximately 1.5 g/L higher biomass within 6 days in modified Murashige and Skoog (MS) medium.
Hydrocarbons and FAMEs
Long-term energy demands will eventually greatly outweigh the world supply of fossil fuels, and their use increases greenhouse gases. Therefore, alternative sources and methods of producing fuels must be found. Although algae can capture greenhouse gas emissions while producing oxygen, the need for high biomass and oil accumulation are challenging for algal-based bioenergy production. S. bacillaris strain siva2011 is rich in lipids, presumably TAGs, with a suitable carbon range for aviation or other liquid fuels. Indeed, scaling studies showed that at 0.2% CO2 supplementation S. bacillaris strain siva2011 had better growth and increased FAMEs in the 8 L bioreactor than 4 L. It is likely that the 20 L bioreactor could have substantially lower hydrodynamic stress. However, further studies on mass transfer at larger scales seem to be warranted. The culture conditions vary from alga species to species. S. bacillaris strain siva2011 can grow in conditions mentioned in this study. Irrespective of the need to further characterize the biochemical pathways for this organism, it is nevertheless important to point out that there is already sufficient empirical evidence that it will likely be a possible candidate for renewable production of light liquid fuels based on the copious production of lipids and hydrocarbons, and especially the relatively high degree of unsaturation found therein.
Materials and methods
Isolation and identification of Stichococcus bacillarisstrain siva2011
Axenic S. bacillaris strain siva2011 cells were isolated from in vitro Lagerstroemia seedlings. The morphology of algal cells was identified by light microscopy. The genus and species were identified by the 18S rDNA region of the nuclear chromosome and the 23S region of the chloroplast rDNA. PCR was performed using primers to amplify the 23S and the 18S region of the rDNA. The products were then sequenced. The genus Stichococcus was identified based on the 18S rDNA sequence in the NCBI database. The identification was confirmed and authenticated by The Culture Collection of Algae at the University of Texas at Austin (UTEX), Austin, TX, USA. The phylogenetic tree was created based on the 18S rDNA sequence using a Clustal X2.0.12 set to exclude positions with gaps, correct for multiple substitutions and run 1,000 bootstrap trials.
The new alga S. bacillaris strain siva2011 was cultured in 5 L and 20 L liquid-phase airlift balloon-type bioreactors [32–34] with modified MS  liquid medium for 6 days. This alga was also tested in BBM for comparison . To evaluate the scale-up potential of the balloon-type bioreactor for larger-scale use, the 5 L bioreactor was used for the 4 L working volume and a 20 L bioreactor was used for the 8 L working volume in order to gain a linear biomass pattern for prediction or modeling. Working volumes of bioreactors for scale-up studies were previously published  for root culture and were not repeated here. Balloon bioreactors have a larger headspace. The 5 L bioreactor has an 8 inch diameter and the 20 L has a 12 inch diameter, which may facilitate efficient light absorption and medium circulation for algal culture. The modified MS medium contains reduced NH4NO3 0.6 g, KNO3 1.5 g [32, 33] with 1% fructose and pH 6.0. The cool white fluorescent room lights at 15 to 30 μE m-2 s-1 for 10 hours followed by 14 hours of dark and 23 to 25°C culture conditions were used. After autoclaving the medium and the bioreactor, the axenic algal cells were cultured into the bioreactor. The inoculum was active cells that were 3 days old and 0.05 g fresh weight (FW)/L. The bioreactor cultures were supplemented with different concentrations of sterile filtered CO2 such as 0.05, 0.1, 0.2 or 0.5%. The input air mixture CO2 gas flow was set at 0.1 vvm (volume (of air) per volume (of liquid) per minute). To screen growth kinetics of S. bacillaris strain siva2011, algal biomass was harvested, and medium pH was measured after 1, 2, 3, 4, 5 and 6 days. Algal cells were harvested by centrifugation at 10,000 rpm for 5 minutes. After harvesting, algal biomass were frozen in liquid nitrogen and freeze-dried. DW was recorded after the samples were freeze-dried to a constant weight.
V1 was base volume at 4 L and V2 was extended at 8 L volume.
Analysis of hydrocarbons and FAMEs
One gram of 6-day-old freeze-dried algal cells were used for analysis of hydrocarbons and FAMEs. The total lipids were evaluated according to Jones et al. . FAMEs were processed according to the AOAC method 996.06 and AOCS method Ce 1 h-05 [39, 40]. Each FAME GC-MS spectra were acquired using a Clarus 500 gas chromatography (PerkinElmer, Waltham, MA, USA) coupled to a Clarus A mass spectrometer (PerkinElmer). A FAMEWAX column (Restek, Bellefonte, PA, USA) was used for separation of FAMEs (30 m length, 0.25 mm ID, 0.25 μm film thickness). The column conditions were determined prior to analysis using a FAME and hydrocarbon reference mixture. Initially, the gas chromatography temperature was 30°C and ramped 10°C/min to a final temp of 220°C and held for 15 minutes at 220°C. Helium was used as the carrier gas. The flow rate was set at 1 mL/min and the spilt ratio was 1:20. The sample injection volume was 1 μL. The mass spectrometer was set to record ranges of spectra from 50 to 500 m/z. The inlet line temperature was set at 300°C and the source temperature was 180°C. Hydrocarbons were processed and analyzed in GC-MS according to Wang et al. . Quantitative analysis of hydrocarbons and FAMEs in the algal biomass was calculated from the calibration curve of the respective standard. Data acquisition and processing were performed with TurboMass software (PerkinElmer).
All experiments were repeated at least three times, each with three replications except sequencing. The experimental variations were expressed as a mean standard error.
Acyl-acyl carrier protein
Bold’s Basal Medium
Fatty acid methyl ester
Free fatty acid
Gas chromatography–mass spectrometry
Murashige and Skoog
Nicotinamide adenine dinucleotide phosphate
National Center for Biotechnology Information
Polymerase chain reaction
The Culture Collection of Algae at the University of Texas at Austin.
This research was funded by the Arkansas Biosciences Institute (grants 262178 and 200109). Part of the mass spectrometry work was supported by the National Institutes of Health (NIH) (P30 GM103450) through the National Institute of General Medicine, and organism identification was supported by the Department of Energy (DOE) (DE-FG36-08BO88036) through the Midsouth/Southeast Bioenergy Consortium. The authors thank Dr David Nobles at UTEX for identification and authentication of S. bacillaris strain siva2011. The technical assistance of Dr Jianfeng Xu, Christopher Easley, Kelsea Brewer, Veronica Hawes and Saritha Kontham (Arkansas State University, Jonesboro, AR, USA), and Dr Jennifer Gidden (University of Arkansas, Fayetteville, AR, USA) was appreciated.
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