Identification and Characteristics of a Cyanobacterium Isolated from a

A photosynthetic species, isolated from an alkaline hot spring in eastern Taiwan, was applied to enhance the dissolved inorganic carbon (DIC) uptake c...
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Environ. Sci. Technol. 2007, 41, 1909-1914

Identification and Characteristics of a Cyanobacterium Isolated from a Hot Spring with Dissolved Inorganic Carbon H S I N T . H S U E H , † H S I N C H U , * ,† A N D CHING C. CHANG‡ Department of Environmental Engineering, Sustained Environmental Research Center, and Institute of Biotechnology, National Cheng Kung University, Tainan 701, Taiwan

A photosynthetic species, isolated from an alkaline hot spring in eastern Taiwan, was applied to enhance the dissolved inorganic carbon (DIC) uptake capability under high alkaline and temperature conditions and was identified. The strain was found to be close to Thermosynechococcus elongatus BP-1 and Synechococcus elongatus based on phylogenetic analysis of 16S rRNA genes. The result was verified with morphological observations. This strain was named as Thermosynechococcus sp. CL-1 (TCL-1). A study of the effects of pH and DIC on the content variations of four elements (C, N, H, and O), lipids (LI), proteins (PR), carbohydrates (CA), and the bioenergy potential was carried out. The concentrations of PR and LI were the highest under the cultivation of pH 7 and CA was at 10.5. According to the analysis of three compositions, the production pathway of LI might be shifted to CA from pH 7 to 10.5 and then shifted to inorganic compounds from pH 10.5 to 11. Regarding the effect of DIC at pH 9, the results revealed that the uptake pathway shift (such as metals uptake) might happen while DIC is less than 18.9 mM. From 18.9 to 47.2 mM DIC, the production pathway of LI shifted to CA and the contents of CA increased quickly from 47.2 to 94.3 mM without a further decrease of LI. Regarding the pyrolysis experiments with a thermogravimetric analyzer coupled with Fourier transform infrared spectroscopy (TG-IR), the transformation of xylan, cellulose, and lignin contents was observed under various pHs and DIC concentrations.

Introduction The Kyoto protocol was based on the obligation of reducing greenhouse gas emissions, aimed especially at lowering the amount of carbon dioxide (1). Carbon dioxide from hot flue gas in thermal sources such as power plants is abundant. Photosynthetic microorganisms not only can remove CO2 from the flue gas but also can produce bioenergy. However, high temperature is a technical barrier in a biological treatment system for CO2 mitigation. Cyanobacteria, such as Synechococcus, which tolerate high temperatures and * Corresponding author phone: (886)-6-208-0108; fax: (886)-6275-2790; e-mail: [email protected]. † Department of Environmental Engineering and Sustained Environmental Research Center. ‡ Institute of Biotechnology. 10.1021/es0620639 CCC: $37.00 Published on Web 02/09/2007

 2007 American Chemical Society

mitigate CO2, could be obtained from hot springs (2). In Taiwan, there are many hot springs and some of their bacteria were isolated to be productive CO2 mitigation tools (3). Other than the light limitation of photosynthesis, carbon dioxide mass transfer was a key factor in cultivating photosynthetic microorganisms due to the low mass transfer coefficient between carbon dioxide and water (4, 5). Therefore, carbon uptake from the carbonate or bicarbonate source in the water might be a better way. A special strain in an alkaline environment bioreactor has been studied in conjuction with a packed tower for absorption of CO2 from flue gas in our lab (4). The results showed that the microorganism was a good candidate. The rationale for this research is to use HCO3-/CO32- as C source to harvest a unique cyanobacterium at high pH and temperature. It was needed to identify and characterize the strain in order to better understand the photosynthetic mechanisms. The morphological approach was one way to show the identity of the strain. However, the molecular approach was sometimes applied simultaneously with the morphological approach to confirm the result (6, 7). The sequences of 16S rRNA genes were usually applied in phylogenetic analyses for evolution based on its conserved properties (6). In this study, the sequences of 16S rRNA genes of some morphologically and physiologically related microorganisms were obtained from the National Center for Biotechnology Information (NCBI) database to analyze their phylogenetic relationship. Since carbon dioxide absorption capacity can be enhanced in alkaline conditions, biomass characteristics (bioenergy) were studied at various pHs and DIC concentrations. The path of carbon transport in the photosynthesis was discovered early (8). Not only were the pathways of external and internal carbon transfer studied intensely in recent years but also related gene expressions (9-11). The role of bicarbonate buffer systems is important for normal physiological function (12). However, the pH increase due to photosynthetic functions under batch cultivation and growth would be inhibited via alkali or the amounts of useful carbon source limitations (13). Commonly, different pH levels would have variations in the ratios of CO2/HCO3-/CO32-. Those variations might change the useful carbon sources due to biological physiology related to carbon-type transporters (11). Therefore, different ratios of C(useful)/N would happen under the same amount of carbon source addition but different pHs. In consequence, those variations might change the biochemical compositions via different pathways under carbon or nitrogen limitations (14). Usually, the variations of macromolecular compositions were focused on four parts, including lipids, proteins, nucleic acids, and carbohydrates (15, 16). A potential method to measure cellular macromolecules was Fourier transform infrared (FTIR) spectroscopy (16, 17). Quantitative analyses have been applied to solid samples via standardized methods (18). Using internal standards such as calcium carbonate or sodium azide was one of the standardized methods (19). Pyrolysis processes were utilized for the commercial production of a wide range of fuels, solvents, chemicals, and other products from biomass feedstocks (20). Thermogravimetric analysis combined with FTIR spectroscopy (TG-IR) could be extended to a large number of volatile pyrolysis products (21, 22). One of the objectives of this study was to determine the properties of the pyrolysis products of biomass cultivated under various pHs and DIC concentrations to understand which cultivation condition would be best for energy production. VOL. 41, NO. 6, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Photoimages of cultivated strain: (a, top left) light microscopy image; (b, top right, and c, lower left) SEM images (B ) section of binary transverse fission); (d, lower right) TEM image (T ) thylakoids).

Materials and Methods Isolation and Cultivation of the Strain Adapted to Alkaline Conditions. Samples of environmental photosynthetic microorganism material were obtained from Chin-Lun (CL) hot spring (pH 9.3, 62 °C) in eastern Taiwan and cultivated in the laboratory. The isolated procedure was based on a serial 5% dilution volume batch suspension cultivation at 50 °C, 2.83 mM, and pH 10-11, with continuous illumination until green materials were found in significant amounts, and then the microorganism was spread on plates to obtain a dominant strain. A modified Fitzgerald medium was adopted as the culture solution (23). The medium was sterilized by an autoclave at 121 °C and 1.5 atm. The cultivation conditions were the same as the isolation stage but with different pHs/ DICs. Regarding the experiments of pH variations, the carbon source was always added as 3 g L-1 Na2CO3 (DIC ) 28.3 mM). Regarding the experiments of DIC variations, the pH levels of the medium were all controlled at 9. The pH value was controlled by automatically adding 0.1 N HCl solution through a feedback system in the constant pH cultivation experiments. Other details of the procedures were documented previously (4). DNA Extraction and Polymerase Chain Reaction Cloning. DNA was extracted from the microorganism following the CTAB-base procedure as described by Stewart and Via (24). We have did at least three independent PCR reactions with two different pairs of primers. One pair of primers (forward primer 5′-AGTTTGATCCTGGCTCAGG-3′ and reverse primer 5′-TACCTTGTTACGACTTCACCC-3′) were designed on the basis of the conserved region in the 16S rRNA gene sequences from 11 species of algae and one species of cyanobacterium (see Supporting Information, Table S1) . Another primer pair is bacterial universal primers (11F, forward primer, 5′-GTTTGATCCTGGCTCAG-3′; 1512R, reverse primer, 5′-GGTTACCTTGTTACGACTT-3′). PCR amplification of 16S rRNA genes was performed in a Thermal Cycle (Hybaid, U.K.) with 5 units of DNA solution, 200 pmol of each primer, 200 nM each deoxyribonucleotide triphosphate, 3 units of Taq DNA polymerase, and 5 µL of 10× Taq DNA polymerase buffer in a 50 µL reaction mixture. The 1910

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amplification was started with one 2-min cycle at 94 °C followed by 35 cycles of 45 s at 94 °C, 45 s at 65 °C, and 2 min at 72 °C. This was followed by one 5-min cycle at 72 °C. The PCR sample was electrophoresed in a 1.2% agarose gel and visualized by staining with ethidium bromide. DNA Sequencing and BLAST Search. Amplified PCR products were purified and directly sequenced. The sequencing reaction was performed by use of a BigDye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA), according to the protocol recommended by the manufacturer. The DNA sequencer was an Applied Biosystems ABI 3700. Database searches were conducted with the BLAST algorithm provided by the NCBI. Phylogenetic Analysis. The DNA sequences of the 16S rRNA gene from 22 species of cyanobacteria were downloaded from GenBank, NCBI, and the target 16S rRNA sequence was fitted into an alignment containing, plus E. coli as an outgroup. All were aligned on the ClustalW website (25). The multiple alignments were refined by eye, and the positions with gaps and undetermined and ambiguous sequences were removed for subsequent phylogenetic analyses. Following the sequence alignment, the neighbor-joining method was used to make the phylogenetic tree with Kimura’s (26) twoparameter distance and resampling statistics of Bootstrap 500 times. This data analysis was carried out with the software package Molecular Evolutionary Genetics Analysis version 2.0 (Mega2). FTIR Analysis. The microorganism sample (10 mg) was mixed with 1000 mg of potassium bromide (KBr) by grinding in a vessel. A portion (200 mg) of this mixture was taken and placed on a pan with a 13 mm diameter and was compressed to a disc shape by an oil pressure machine. CaCO3 (an internal standard; 0.5 mg) was mixed with 199.5 mg of KBr and compressed to a disc shape, following the same procedure. For the quantitative analysis, spectra were obtained on an FTIR spectrometer (Perkin-Elmer Spectrum One): from 4000 to 450 cm-1, transmittance type of spectra, a resolution of 2 cm-1, a scan speed of 0.2 cm s-1, and scans of 100 duplications. The procedure was repeated three times.

FIGURE 2. Distance tree based on partial 16S rRNA sequence analysis of the cultivated strain named as Thermosynechococcus sp. CL-1, and other complete and partial cyanobacterial sequences published in the NCBI database. E. coli (also published in the NCBI database) was used for rooting the tree. Numbers at nodes indicate the numbers of differences (from the cluster descending from the node) found in the 500 bootstrap trees. Clusters I and II, unicellular and colonial forms lacking specialized cells or reproduction; cluster III, others. Thermogravimetric Analysis. The biomass samples cultivated at various pHs and DIC concentrations were used in the pyrolysis study with TGA. The TG-IR instrument was used for the analysis of evolved gases. In this process, as the sample was heated, the evolving volatile products were carried out of the furnace directly into a gas cell with a 5 cm diameter and 15 cm of optical path length (controlled temperature is 200 °C). The temperature of the gas tube between TGA and FTIR was controlled at 250 °C by a heating instrument to avoid the condensation of volatiles. For TGA programming, 5-15 mg of biomass materials was heated in nitrogen at 10 °C min-1, first to 150 °C to dry for 4 min and then to 900 °C for pyrolysis.

Results and Discussion Identification of Isolated Microorganism from a Hot Spring by 16S rRNA Gene Analysis and Morphology. The 16S rRNA gene is a conserved DNA sequence and was used as a molecular marker for phylogenetic classification in microorganisms. In the beginning of our experiment, we knew only that our target species was photosynthetic; whether it was a eukaryotic alga or a cyanobacterium was not clear. Therefore, in order to do phylogenetic analysis, the PCR primer was designed on the basis of conserved region of 16S rRNA genes among 11 species of algae and a species of cyanobacterium (see Supporting Information, Table S1). Following the procedure of PCR and sequencing, we obtained an identical sequence with 1353 bps from at least three independent experiments. The results of BLAST in NCBI

revealed that the sequence was very close to that of Thermosynechococcus elongtus BP-1 and Synechococcus elongatus genes, with a shared identity of 99% (see Supporting Information, Table S2). In addition to these two species, five others also belonged to the Synechococcus genus. To confirm that the cultivated microorganism belonged to the Synechococcus genus, which is a bacterium, therefore, we used one pair of universal primers, 1512R and 11F, to redo the PCR. The results were similar to the previous (see Supporting Information, Table S2). The UV-vis spectrum were measured in the cultivated target strain. The peak wavelengths (403, 437, 487, 588, 625, 664, and 678 nm) (see Supporting Information, Figure S1) were approaching those of cyanobacteria and were different from those of diatoms and green algae as described in a previous report (27). Rippka et al. (28) divided the cyanobacteria into five sections. Section I specified unicellular cyanobacteria that reproduced by binary fission or budding. A subsection of section I specified cylindrical to ovoid cells that reproduced by binary transverse fission. There were three genera, Synechococcus, Gloeothece, and Gleobater, in this subsection. According to our observations under light microscopy and SEM, Figure 1a,b showed that this strain was unicellular with oval shape, and cell division occurring by binary transverse fission (Figure 1c). The size of cells was 1-2 µm wide and 5-10 µm long. The morphological features were very similar to the characteristics of this subsection. In addition, the different types of thylakoid arrangements could also provide additional information for taxonomical assignVOL. 41, NO. 6, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Specific normalized FTIR spectral area variations (LI, PR, and CA) of biomass under various pH cultivations and a fixed DIC at 28.3 mM. ment in cyanobacteria. From our observations by TEM, the intracellular ultrastructural pattern for the arrangement of thylakoids in this strain was coccoid types (Figure 1d). According to a previous report, coccoid-type arrangement of thylakoids was indeed an apparent characteristic of genus Synechococcus (29). Therefore, the morphological features of this target strain are identical to those of Synechococcus genus. The phylogeny of this isolated strain was further determined. Besides the cultivated isolated strain, 22 other cyanobacterial species including 13 species of genus Synechococcus and an outgroup, E. coli, were chosen from the different morphologies illustrated in Figure 2. Their 16S rRNA

genes were obtained from the NCBI database, and E. coli was used for rooting the tree. The phylogenetic tree suggested that the species could be clustered into three groups (Figure 2). In addition, our cultivated strain could be clustered with species of Synechococcus elongatus and Thermosynechococcus elongatus and separated from other species of Synechococcus (Figure 2). However, the thermophilic cyanobacterium Thermosynechococcus elongatus strain BP-1 was derived from a hot spring in Beppu, Japan. It previously had been identified as Synechococcus elongatus on the basis of cell morphology (30). However, this cyanobacterium was distantly diverged from all other Synechococcus clusters based on the analysis of 16S rRNA gene (31). Therefore, Katoh et al. (32) tentatively renamed it as Thermosynechococcus elongatus, which is defined for mesophilic species derived from fresh water. Because the strain we isolated was also a mesophilic one, we tentatively named it as Thermosynechococcus sp. CL-1 (CL was an abbreviation from Chin-Lun hot spring). That followed recommendation 12c of Bacteriological Code, “Choose a specific epithet that, in general, gives some indication of a property of the source or of the species”. Elements and IR-Spectroscopic Quantitative Analysis of Biomass Cultivated under Various Conditions. Four Fourier transform infrared (FTIR) spectrum peaks, including 2924 (PI), 2509 (PII), 1650 (PIII), and 1053 (PIV) cm-1, were simulated by curve fitting and the areas under the peaks were taken as the quantitative analysis of LI, internal standard, PR, and CA, respectively. Three spectral peaks (PI, PIII, and PIV) were almost the same before (shown in Supporting Information, Figure S2a) and after addition of the internal standard [CaCO3 (PII)] (shown in Supporting Information, Figure S2b) and could be taken as the characteristic peaks of LI, PR, and CA, respectively (see Supporting Information,

FIGURE 4. Variations at various DIC concentrations and a fixed pH at 9: (a) growth rate; (b) content of LI, PR, and CA; (c) content of N, H, C, and O. 1912

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Table S3). In Supporting Information, Figure S3b,d,f, simulated absorption spectra using several Gauss curves were obtained according to the fourth-derivative curves in Figure S3a,c,e. The content variations of LI, PR, and CA in the biomass at various pHs and a fixed 28.3 mM DIC concentration could be expressed based on the relative peak areas (PI/PII, PIII/PII, and PIV/PII) as shown in Figure 3. From Figure 3, the concentration of PR and LI were highest under the cultivation of pH 7, and the highest CA was at 10.5. However, no variations were observed regarding the contents of C, H, and O between pHs 7, 9, and 10.5 (4). This phenomenon could be explained by the effect of different uptake pathways of DIC types, including CO2, HCO3-, and CO32-. In addition, the contents of C and N were highest at pH 11 but PR, LI, and CA were not. This result could be explained by more inorganic compounds produced at such a high pH, such as white event (33). In brief, from pH 7 to 11, the production pathway of LI shifted to CA from pH 7 to 10.5 and then shifted to inorganic compounds from pH 10.5 to 11. The growth rates were almost the same from pH 7 to 10.5 and declined at pH 11 in a previous study (4). LI and CA production pathways might not affect the growth rates, but the inorganic compounds did. A study of specific growth rates at various DIC concentrations and a fixed pH of 9 was carried out. The growth rates increased gently with an increasing DIC concentration from 4.7 to 94.3 mM (Figure 4a). Although no significant difference of growth rate was observed at 4.7 mM compared with other DIC concentrations, the contents of LI, PR, and CA at 4.7 mM were all the lowest (Figure 4b). The results for C, N, H, and O contents were similar to those for LI, PR, and CA (Figure 4c). As shown in Figure 4b, the contents of LI, PR, and CA all increased with DIC concentrations below 18.9 mM. These results reveal the possibility of different uptake pathways (such as metals uptake shown in Supporting Information, Figure S4) at such low DIC concentrations. From 18.9 to 47.2 mM DIC concentrations, the production pathway of LI shifted to CA. From 47.2 to 94.3 mM, the contents of CA increased quickly without further decreasing LI.

FIGURE 5. Weight loss in specific temperature ranges under various conditions: (a) Varied pH at a fixed DIC of 28.3 mM; (b) varied DIC concentration at a fixed pH of 9. R1, 220-300 °C; R2, 300-380 °C; R3, >380 °C; total, R1 + R2 + R3.

Pyrolysis Analysis of Biomass by TG-IR. Pyrolysis experiments of biomass cultivated at various pHs and DIC concentrations were carried out. The thermal weight losses, which ranged from 220 to 900 °C, could be divided into three regions (R1, 220-300 °C; R2, 300-380 °C; and R3, >380 °C) according to the major thermal weight losses of pyrolysis of xylan (semicellulose) (R1), cellulose (R2), and lignin (R3) (shown in Supporting Information, Figure S5). As shown in Figure 5a, the losses of R1 and R3 in the biomass were the highest when cultivated at pH 9, while R2 was the highest at pH 10.5. This result also matched with the case of the most CA at pH 10.5. In others words, carbohydrates were accumulated at pH 10.5 as cellulose. By pyrolysis of the biomass grown at various DIC concentrations and the fixed pH 9 cultivations, the lowest weight loss of R1, R2, and total weight loss were observed at 4.7 mM DIC (Figure 5b). Below 18.9 mM, the weight loss of R2 increased with an increase of DIC concentration but then decreased after this concentration. At the same time, the weight loss of R1 increased quickly with an increase of DIC concentration below 9.4 mM but gently above this concentration. In other words, the weight losses of R1 and R2 increasing below 9.4 mM DIC might be due to the shift of a pathway from C/N uptake to some mechanisms such as metals uptake (shown in Supporting Information, Figure S4) and the shift of cellulose production to xylan production. The content of residues at the final pyrolysis temperature (900 °C) was 22% for the case of 4.7 mM DIC. This content was much more than that for the case of 94.3 mM (14%). This result also revealed that a different metabolism pathway at

FIGURE 6. TG-IR spectrum ranging from 3050 to 2800 cm-1 in the temperature range from 273 to 685 °C: (a) CH4; (b) CH3 asymmetrical stretch; (c) CH2 asymmetrical stretch; (d) CH3 symmetrical stretch. such a low DIC concentration, such as metals uptake, might happen. As for IR analysis for the evolved gases from TGA, CH4, CO2, CO, H2O, and some evolution of hydrocarbons were found in various temperature ranges (shown in Supporting Information, Figure S6). The gas products of hydrocarbons evolved mainly from 280 to 380 °C (Figure 6). This range covered the pyrolysis temperature of xylan and cellulose. In addition, some IR spectra of hydrocarbons were also found in the range of 1200-900 cm-1 in the same temperature range (shown in Supporting Information, Figure S6).CH4 started to evolve above 380 °C and ended at about 680 °C. Therefore, VOL. 41, NO. 6, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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liquefied fuel might be produced from 280 to 380 °C and gasified fuel might be produced from 380 to 680 °C.

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Acknowledgments We gratefully acknowledge the National Science Council, Republic of China, for their financial support (NSC94-2211E-006-086).

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Supporting Information Available

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Information about UV-vis and FTIR spectra of the biomass, metal and organic compositions of the biomass,and the strain with 16S rRNA for designing primers. This material is available free of charge via the Internet at http://pubs.acs.org.

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Received for review August 29, 2006. Revised manuscript received December 26, 2006. Accepted January 3, 2007. ES0620639