ISSN 10214437, Russian Journal of Plant Physiology, 2013, Vol. 60, No. 4, pp. 549–554. © Pleiades Publishing, Ltd., 2013.
RESEARCH PAPERS
Evaluating New Isolates of Microalgae from Kazakhstan for Biodiesel Production1 Y. M. Dyoa, S. E. Vonlanthenb, S. Purtonb, and B. K. Zayadana a
Department of Biotechnology, AlFarabi Kazakh National University, 71, AlFarabi ave, 050040, Almaty, Kazakhstan b Institute of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, United Kingdom; fax: +44 (0)20 76797193; email:
[email protected] Received October 30, 2012
Abstract—New microalgal strains that are native to SouthEast Kazakhstan were isolated and characterized with a view to identifying suitable candidates for biodiesel production. Six strains of chlorophyte algae (named K1–K6) were recovered from environmental samples as axenic cultures, and molecular analysis revealed that five (K1–K5) are strains of Parachlorella kessleri, whereas K6 is a strain of Chlorella vulgaris. A third isolate from Uzbekistan (termed UZ) was also identified as a separate strain of P. kessleri. All strains show high growth rates and an ability to utilize acetate as an exogenous source of fixed carbon. Furthermore, under conditions of nitrogen depletion, all three strains showed a significant accumulation of neutral lipids (triacylglycerides). P. kessleri K5 and C. vulgaris K6 therefore represent promising autochthon strains for largescale cultivation and biodiesel production in Kazakhstan. Keywords: Chlorella vulgaris, Parachlorella kessleri, biodiesel, lipids DOI: 10.1134/S1021443713040031 1
INTRODUCTION
Microalgae comprise a genetically and ecologically diverse group of photosynthetic microorganisms that account for ~50% of global organic carbon fixation [1], and in the search for renewable and carbonneu tral alternatives to fossil fuels, there is increasing inter est in the exploitation of suitable microalgal species as a feedstock for lipid extraction and conversion to biodiesel [2, 3]. Unlike the oleaginous crops that are currently cultivated for biodiesel, microalgae could be cultivated at industrial scale on nonarable, lowgrade land using wastewater or seawater and could be cou pled to CO2 sequestration. Furthermore, the photo synthetic efficiency, the high content of storage lipids, and fast growth rates of some species offer the promise of high and continuous lipid productivity [4]. How ever, the current cost of algal biodiesel production is too expensive to be competitive with either conven tional diesel or plantderived biodiesel. This is due in part to the challenge of sustainable cultivation in industrialscale open systems, in which the algae have to be capable of tolerating the fluctuations in local cli matic conditions; have to be sufficiently robust to out 1 This text is published in original.
Abbreviations: ITS—internal transcribed spacer; PCR—poly merase chain reaction; TAGs—triacylglycerides; TAP—Tris acetatephosphate.
compete other invasive species, and must be able to survive numerous predators and pathogens. In addi tion, the accumulation of large amounts of neutral lip ids (TAGs: triacylglycerides) in most algae is only seen under conditions of nutrient stress where growth is compromised [5]. Consequently, production involves the optimization of two phases – maximal growth to produce biomass, and then a period of stress (e.g. by depletion of fixed nitrogen in the medium) to induce lightdriven biosynthesis and accumulation of TAGs within the algal biomass. Initial strain selection for a chosen geographical region is perhaps best achieved by surveying indige nous algae that are already adapted to the local envi ronment and to the seasonal climatic patterns. Prom ising strains can then be used as the basis for genetic improvements or “domestication”, using mutation and selection approaches, or possibly genetic engi neering [5]. Currently, the major focus is on several genera of marine microalgae, including Nannochlo ropsis, Tetraselmis, and various diatoms, and a few freshwater genera, such as Chlorella, that show high lipid productivity [4, 6]. As a first step towards identi fying suitable microalgal strains for biodiesel produc tion in Kazakhstan, we report the isolation and char acterization of strains of chlorophyte algae from envi ronmental samples collected in the mountains near the city of Almaty. Two axenic strains are described
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Fig. 1. Brightfield microscope images of K5, K6, and UZ strains grown in nitrogenreplete medium.
that show high growth rates and good TAG accumula tion under nitrogen stress. One is identified as a new strain of Parachlorella kessleri and the other a strain of Chlorella vulgaris. MATERIALS AND METHODS Soil and water samples were collected in Septem ber 2011 from the Zailiysky Alatau gorge in the Tian Shan mountain range south of Almaty, Kazakhstan, and used to inoculate liquid Tamiya medium [7] in 10 mL translucent sample bottles. After three weeks of incubation at room temperature and in natural light, aliquots were plated by streaking on Petri dishes con taining solid Tamiya medium. Green material that appeared following several weeks of incubation in the light was restreaked to fresh medium containing the antibiotic ampicillin (100 µg/mL) to inhibit the growth of contaminating bacteria. Isolated green col onies were picked and restreaked to fresh medium, and their axenic state was determined by plating on the rich medium, Meat Peptone Agar, to encourage bacte rial growth [8]. Brightfield and fluorescence micros copy was carried out using an EVOS fl digital fluorescent microscope (PEQLAB, Germany). For the fluorescence imaging, the microscope was fitted with an RFP light cube (excitation 531/40 nm) and a 593/40 nm emission filter. For molecular analysis, total genomic DNA was isolated from single fresh colonies, using the Chelex method described in [9], in which cells are lysed in 50% ethanol and cell debris is removed by the addition of 5% Chelex100 resin, followed by boiling for 5 min, brief centrifuging, and final recovering the supernatant containing DNA. For PCR analysis, 2 µL of the supernatant was used in a standard 25cycle reaction using Phusion DNA polymerase (New England Biolabs, United States) according to the manufac turer’s recommendation. Primer pairs used were as recommended by Hall et al. [10] as follows: rbcL.M28F and rbcL.M1390R; tufA.F and tufA.R; ITS1 and ITS4. PCR products were purified using
ionexchange spin columns (Qiagen, Germany), quantified using a Nanodrop spectrometer, and sent for commercial sequence determination with the for ward primer (rbcL.F or tufA.F) as the sequencing primer. DNA sequence was processed using MacVec tor software and used in phylogenic analysis with cor responding rbcL or tufA sequences obtained by the Purton lab from known chlorophyte species or down loaded from the GenBank nucleotide database (www.ncbi.nlm.nih.gov). Alignment and phylogram construction was carried out using CLUSTALW2 (www.ebi.ac.uk/Tools/msa/clustalw2). Growth and lipid analysis were carried out by growing the strains in liquid TAP (Tris–acetate–phos phate) medium [11] at 25°C and continuous white light of approximately 50 µmol/(m2 s), with gentle agi tation (100 rpm). For induction of lipid accumulation, +
the concentration of nitrogen (in the form of N H 4 ) was reduced tenfold from 7.48 mM (nitrogenreplete TAP) to 0.748 mM (nitrogenlimited TAP). Growth was assayed by measuring the light scattering of the culture at 750 nm using a UNICAM Spectrometer UV2. For growth tests on solid medium, mixotrophic and heterotrophic growth was tested on TAP medium that contained 17.4 mM acetate as a carbon source; whereas phototrophic growth was on medium lacking acetate. For mixotrophic and phototrophic growth tests, incubation was under continuous light of 50 µmol/(m2 s) at 25°C for 3 days; for heterotrophic growth tests the dishes were wrapped in aluminum foil. Lipid accumulation under nitrogendepleted con ditions was assessed by fluorescence spectroscopy using the fluorescent dye, Nile red [12]. Strains were grown for five days in nitrogenreplete and nitrogen limited TAP. Nile red staining was performed using Nile red at a final concentration of 2 µg/mL in 25% (v/v) DMSO to allow penetration of the dye through the cell wall. Fluorescence was measured using a Per kinElmer LS55 Luminescence Spectrometer with
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Haematococcus pluvialis Dunaliella salina CCAP 19/18 Chlamydomonas reinhardtii Oltmannsiellopsis viridis Pyramimonas parkeae Uzbekistan isolate UZ Parachlorella kessleri SAG 2111 lg Kazakhstan isolate K5 Chlorella sorokiniana UTEX 1230 Chlorella sorokiniana H1983 Chlorella vulgaris C2 Chlorella vulgaris CCAP 211/11B Kazakhstan isolate K6 Chlorella variablis (b) Pedinomonas minor Oltmannsiellopsis viridis Dunaliella salina CCAP 19/18 Coccomyxa sp. C169 Trebouxia aggregata SAG 2191d Parachlorella kessleri SAG 21111g Kazakhstan isolate K5 Chlorella vulgaris Chlorella sorokiniana Pyramimonas parkeae Chlamydomonas reinhardtii Fig. 2. Molecular phylogeny of strains K5, K6, and UZ. DNA sequence derived from the chloroplast genes tufA (a) or rbcL (b) were aligned using CLUSTALW2 with equivalent sequences for known species of chlorophyte algae. Note: K6 and UZ failed to produce rbcL PCR product.
the excitation wavelength set at 510 nm and the emis sion scanned between 520 and 800 nm. RESULTS AND DISCUSSION Environmental samples of soil, snow, and stream water were collected from a site in the Tian Shan mountains near Almaty. From one of these samples we were able to obtain six axenic strains of green algae, named K1 to K6. Bright field microscopy revealed that all six strains were nonflagellated spherical cells of approximately 3–5 microns in diameter (Fig. 1), with K6 smaller that K5 (K1–K4 being identical to K5 – see below), and both isolates smaller than a third strain previously obtained from an environmental sample from Uzbekistan (strain UZ). Molecular identification of the isolates was carried out by PCR amplification and DNA sequencing, using three pairs of primers for the nuclear ITS2 rDNA region and the chloroplast genes rbcL and tufA [10]. For K1 to K5, good sequence was obtained for rbcL and tufA, but multiple overlapping sequences were obtained from the ITS2 PCR product suggesting poly morphisms within the genomic ITS2 regions. Align ment of both sets of chloroplast sequences revealed a RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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100% match in both cases, confirming that all five iso lates are the same strain. K5 was therefore chosen as the representative strain. For K6 and UZ, good ITS2 sequence was obtained, but no PCR product could be generated using the rbcL primers, suggesting that sequence variation in one of the primerbinding regions was preventing efficient priming of DNA syn thesis. BLASTn searches of the GenBank database using the DNA sequences, and subsequent alignment K5 K6 UZ photo mixo hetero
Fig. 3. Growth of K5, K6, and UZ on solid medium under different trophic conditions. Phototrophic growth = light without exogenous acetate; mixotrophic growth = light with acetate; heterotrophic growth = no light, but exogenous acetate. No. 4
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Fig. 4. Growth of K5, K6, and UZ in medium containing either replete nitrogen (100% N) (solid lines) or limited nitrogen (10% N) (dotted lines). Growth of each culture was monitored by measuring the optical density at 750 nm. —䊉— K5 —䊊— K6 —䉭— UZ 䊉 K5 10% N 䊊 K6 10% N 䉭 UZ 10% N
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of matching sequences from other green algal species, revealed that K5 and UZ are different strains of P. kessleri, whereas K6 is a strain of C. vulgaris. Phylo genetic trees obtained from the tufA and rbcL sequences are shown in Fig. 2. Many species of Chlorella are capable of het erotrophic or mixotrophic growth utilizing an exoge nous carbon source, such as glucose or acetate, and cultivation under such conditions can influence lipid productivity [13]. In order to determine whether K5, K6 and UZ could utilize a fixed carbon source, the strains were tested for mixotrophic and heterotrophic growth on media containing acetate. As shown in Fig. 3, K5, K6 and UZ are all capable of using acetate and grow faster in the light when acetate is available. In darkness, K5 and UZ grow at a similar rate to pho totrophic growth, but K6 heterotrophic growth is lim ited. Further growth analysis was carried out by cultur ing the three strains under mixotrophic conditions in medium containing either replete levels of nitrogen (as +
N H 4 ) or 10% of this level, such that nitrogen became depleted during the growth. As shown in Fig. 4, growth rates were similar for all three strains with a calculated doubling time of approximately 11 h, although K6 showed a longer lag phase. However, in the late expo nential phase (around 80–100 h), it is clear that nitro gen became depleted in the 10% N medium and growth slowed relative to the 100% N cultures, so that final cell densities were markedly lower (Fig. 4).
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Fig. 5. Fluorescence spectra from Nile red staining of K5, K6, and UZ after five days of cultivation in nitrogen replete (lower line) and nitrogen limited (top lines) media. Staining was performed using 25% DMSO to allow pene tration of the dye through the cell wall. Excitation wave length was 510 nm and emission was scanned between 520 and 800 nm. TAGs show a peak at 580 nm for P. kessleri strains K5 and UZ and 574 nm for C. vulgaris strain K6. A second peak of DMSO/Nile red fluorescence is at 650 nm, and chlorophyll fluorescence is seen at 690 nm.
The accumulation of neutral lipids (triacylglycer ides; TAGs) was demonstrated by the treatment of the cell suspensions, after 120 h of growth as above, with the lipophilic fluorescent dye, Nile red [12]. As seen in the fluorescence spectra (Fig. 5), the Ndepleted cul tures showed a marked accumulation of TAGs in all three strains. However, it should be noted that this assay is only semiquantitative and direct comparison of lipid levels between the three species is not possible [14]. The accumulation of large lipid bodies within the cells under nitrogen stress conditions was confirmed by fluorescence microscopy, as shown for the UZ strain in Fig. 6. Further analysis of the lipid composition and the lipid productivity of K5 and K6 under a range of
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Fig. 6. Visualization of storage lipid accumulation within cells. UZ strain grown in nitrogenreplete (a, b) and nitrogen limited medium (c, d). Bright field images (a and c) and Nile red stained cells (b and d) are visualized by fluorescence microscopy.
growth conditions is now required to determine whether these represent suitable autochthon strains for largescale cultivation in Kazakhstan with a view to biodiesel production. ACKNOWLEDGMENTS Y.M.D. was supported by a scholarship from the AlFarabi Kazakh National University, and S.E.V. by an industrial CASE studentship from the U.K. Bio technology and Biological Sciences Research Council in partnership with Syngenta. Algal lipid research in the Purton Group is supported by the E.U. Frame work Program 7 project GIAVAP (KBBE 20103: GA266401). REFERENCES 1. Field, C.B., Behrenfeld, M.J., Randerson, J.T., and Falkowski, P., Primary production of the biosphere: integrating terrestrial and oceanic components, Sci ence, 1998, vol. 281, pp. 237–240. RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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