3.2. DNA Amplification, Purification, Cloning and Sequencing
The oligonucleotide primers used to amplify both the cyanobacterial 16S rDNA and the microalgal chloroplast 16S rDNA were CYA106F, CYA359F and CYA781R [18
]. In addition, the universal bacterial primer 1541R (5′ AAGGAGGTGATCCAGCC 3′) was employed to obtain longer sequences of the cyanobacterial 16S rRNA genes.
The Polymerase Chain Reaction (PCR) assays were performed as described elsewhere [17
], using the following profile: 40 cycles of 94 °C for 1 min, 50 °C for 1 min and 72 °C for 1 min, followed by an extension at 72 °C for 7 min. Visualization of the amplified DNA products was performed using 1.5% agarose gel electrophoresis. DNA fragments were isolated from agarose gels using the GFX PCR-DNA and Gel Band Purification kit (GE Healthcare, UK), according to the manufacturer’s instructions. The purified PCR products were cloned into the pGEM®
-T Easy vector (Promega Corporation, Madison, WI, USA), and further used to transform E. coli
DH5α competent cells—following again the manufacturer’s instructions. Colonies were screened for presence of the insert via colony-PCR, and subsequently grown overnight in liquid LB medium at 37 °C with shaking. Plasmid DNA was isolated using the GenEluteTM Plasmid Miniprep Kit (Sigma-Aldrich, St. Louis, MO, USA), and sequencing was performed at STAB Vida (Lisbon, Portugal). Computer-assisted sequence analysis and comparisons were performed using Vector NTI Advance 10 (Invitrogen Corporation, Carlsbad, CA, USA). Novel sequences associated with this study are available from GenBank, under the accession numbers depicted in Table 5
List of GenBank accession numbers of novel 16S rDNA sequences obtained, and closest cultured relatives.
List of GenBank accession numbers of novel 16S rDNA sequences obtained, and closest cultured relatives.
|Isolate||GenBank Accession Number||Closest Cultured Relative (% similarity, accession number) a|
|J52||EU073188||Anabaena planctonica strain 71 (99%, AJ293108)|
|M2-7||EF634458||Limnothrix sp. strain CENA110 (99%, EF088338)|
|M2-1||EU073189||Scenedesmus obliquus strain UTEX 393 (98%, DQ396875)|
|M2-6||EU073191||S. obliquus strain UTEX 393 (92%, DQ396875)|
|M3-9||EU073193||S. obliquus strain UTEX 393 (92%, DQ396875)|
|M4-3||EU073194||S. obliquus strain UTEX 393 (92%, DQ396875)|
|M4-5||EU073195||S. obliquus strain UTEX 393 (92%, DQ396875)|
|M2-5||EU073190||S. obliquus strain UTEX 393 (99%, DQ396875) |
|M2-18||EU073192||S. obliquus strain UTEX 393 (99%, DQ396875)|
3.3. Growth Conditions and Microorganisms
Environmental isolates and culture collection species/strains tested in this study are listed in Table 1
for cyanobacteria, and in Table 2
Batch cultures were grown at 25 °C, in 200 mL of medium, under continuous illumination with fluorescent daylight (35 μmolphoton
), using preferentially OHM [20
] and BG11/BG110
] media, for microalgae and cyanobacteria, respectively. For marine isolates, ASW [22
] and ASW-BG11 [23
] media were used instead.
Genomic cyanobacterial DNA and total microalgal DNA were extracted according to Tamagnini et al.
] and Burja et al.
]. DNA amplification, purification, cloning and sequencing was performed at STAB Vida (Lisbon, Portugal); computer-assisted sequence analysis and comparisons were performed using Vector NTI Advance 10 (Invitrogen Corporation, Carlsbad, CA, USA). Novel sequences associated with this study were made available from GenBank.
3.4. Extracellular and Intracellular Extraction
Extracellular and intracellular extracts were collected according to Guedes et al.
]. In short, 5 mL of a 30 day-old culture was centrifuged at 4000 rpm, for 7 min at 15 °C, and the supernatant (i.e.
, extracellular extract) was collected. The pellet was then resuspended and homogenized in 5 mL of a mixture of ethanol and water (1:1 v/v); the cells were crushed in an Ultra Turrax T 18 homogenizer (Ika, Wilmington, NC, USA), at 14,000 rpm for 30 s, and then subjected to centrifugation at 4000 rpm, for 5 min at 15 °C; finally, the supernatant (i.e.
, the intracellular extract) was collected.
3.5. Chlorophyll a Content
The total chlorophyll a
content was determined after extraction of the cyanobacterial cells using 90%(v/v) methanol; absorbance was measured at 663 nm, and the equation μgchlorophyll a
= 12.7 × Abs663nm
—previously proposed by Meeks and Castenholz [25
], was taken advantage of for quantification as reference basis.
Although a per biomass weight basis would in principle have been preferable because secondary rather than primary production was at stake, parallel determination of the content of chlorophyll a per dry biomass of a few species taken at random from each genus would not indeed alter the decision on selection of strains throughout screening, nor would it reverse any major conclusion; this is so because there is a strong correlation between dry weight and chlorophyll a content. The basis chosen is not only easier to quantitate, but also allows more meaningful discussion vis a vis with available data in the literature (owing to it being a regular trophic indicator). The known dependence of chlorophyll a levels on the physiological status of the cells did not apparently interfere with the figures generated, as in our case the cultures were all in their early stationary phase when harvested.
3.6. ABTS Scavenging Assay
The radical-scavenging capacities of both extra- and intra-cellular extracts of microalgae and cyanobacteria were evaluated via the ABTS radical cation (ABTS•+
) assay, as originally detailed by Re et al.
] and recently refined by Gião et al.
]—using, in the case of extracellular extracts, plain growth medium as control. The cation ABTS•+
was diluted with ultra-pure water, for extracellular extracts; and in a mixture of ethanol and water (1:1 v/v), for intracellular extracts; hence full quantification of both water- and lipid-soluble antioxidants was possible. The results were expressed in Antioxidant Activity Units (AAU), where 1 AAU is defined as 1 mg L−1
of equivalent ascorbic acid per μg of chlorophyll a
3.7. Deoxyribose Protection Assay
The deoxyribose protection was quantified via a method described in detail elsewhere [28
]. In brief, a 200 μL-sample of the extract of interest was added to 10 μL of 100 mmol L−1
deoxyribose, and incubated for 1 h at 37 °C—in the presence of 10 μL of 10 mmol L−1
, 10 μL of 1 mmol L−1
33% (w/v) H2
, and 10 μL of 10 mmol L−1
EDTA, in a 24 mmol L−1
sodium phosphate buffer (pH 7.4) containing 15 mmol L−1
NaCl, to generate hydroxyl radicals.
The aforementioned radicals break deoxyribose into fragments which, in the presence of 1 mL of 1% (v/v) TBA in 0.05 mol L−1 NaOH, under acidic conditions (i.e., 1.5 mL of 28% (v/v) tricloroacetic acid) and high temperature (i.e., 100 °C for 15 min), give rise to a chromophore (malonaldehyde); this adduct was quantified by absorbance at 532 nm, using a Heλios α spectrophotometer (Unicam, Cambridge, UK). When antioxidants are present, they compete for hydroxyl radicals, thus decreasing the extent of fragmentation of deoxyribose.
All measurements were made against adequate blanks; and triplicate samples were taken, with analyses run in quadruplicate for each one.
3.8. DNA Protection Assay
Cell-free microalga extracts were prepared using 400 mg of lyophilized culture, resuspended in 12 mL of ethanol/water (1:1, v/v) and disrupted by ultrasonication for 15 min. Their protective effects against DNA oxidative damage induced by Cu(II)-ascorbic acid were assessed following Muñiz et al.
] and Perez et al.
]. For that purpose, each reaction mixture was prepared so as to contain 50 μg of calf thymus DNA, 10 mM of ascorbic acid and 100 μM of Cu(II), as well as various volumes of cell-free extract (i.e.
, 50, 75, 100, 150 and 200 μL); the final volume was in all cases adjusted to 1 mL with 100 mM sodium phosphate buffer (pH 7.4).
The reaction mixtures were incubated in a shaking water bath, at 37 °C for 1 h, and 20 μL of each was loaded onto a 1.1% (w/v) agarose gel. Electrophoresis was performed according to the protocol described by Sambrook and Russell [19
], using 1× TAE buffer; and DNA was visualized using ethidium bromide, under UV light.
3.9. Bacteriophage Protection Assay
Cell-free microalga extracts were prepared using 250 mg of lyophilized culture, resuspended in 5 mL of a mixture of ethanol/water (1:1, v/v), followed by cell disruption via sonication for 15 min, and final stirring for 10 min; the sample was then filtered through a sterile filter (0.22 μm). Details of this method were described previously [12
The rationale for this methodology is a virucidal attack on Salmonella Typhimurium cells; when a challenging oxidant (250 mM H2O2) is initially applied, the bacteriophage infection capacity decreases; however, if protection occurs in the presence of antioxidant compound(s), the virus will recover its normal infection capacity.
More specifically, the stocks of phage P22 and Salmonella Typhimurium (ATCC 19585 P1) were prepared according to ATCC indications. Dilutions of the virus up to 10–12 were performed (in duplicate) in tryptone soy broth (TSB, from LAB M); 100 μL of each dilution was then mixed with 300 μL of Salmonella culture, harvested in the exponential phase—i.e., a 1% (v/v) inoculum of an overnight culture in TSB at 37 °C, into fresh TSB was incubated at 37 °C for a further 2.5 h. Infection was allowed to proceed for 10 min, in a water bath kept at 37 °C; then, 100 μL was spread onto pre-dried plates of tryptone soy agar (TSA, from Biokar Diagnostic), and incubated at 37 °C for 24 h. Following incubation, the plaques were counted and the total viable phage numbers were accordingly calculated (and expressed in plaque-forming units per unit volume, PFU mL−1); the higher this figure, the stronger the infection capacity of the virus. Each mixture was left to react for 20 min, at room temperature (in duplicate), and was then quenched via addition of 50 μL of 500 U mL−1 catalase (Sigma). Aliquots of 100 μL were collected every 5 min for a period of 20 min, and serial decimal dilutions were performed up to 10−6.
For the infection stage, 100 μL of each phage dilution was added at a time to 300 μL of the suspension of Salmonella in the exponential phase (as described above), and incubated at 37 °C for 10 min. Then, 100 μL was spread onto pre-dried TSA plates (in duplicate), and incubated at 37 °C for 24 h; the viable phage number was again expressed as PFU mL−1. The difference between the viable phage numbers in the presence of sample and oxidant (SOP), and only in the presence of oxidant (OP), were considered as a datum point; when these were positive, then samples exhibited an antioxidant effect, and negative values indicated obviously otherwise.
3.10. Antioxidant Identification
Microalgal cell-free extracts were prepared using 40 mg of lyophilized culture, resuspended in 5 mL of acetone/hexane (1:1, v/v), and disrupted via ultrasonication for 15 min. Full description of the HPLC procedure is available elsewhere [24
]; neoxanthin, violaxanthin, lutein, zeaxanthin, and β-carotene were assayed for, owing to their known antioxidant features.
Although the type of compounds extracted depends in general on the nature of the extracting solvent(s), water was not allowed in the aforementioned HPLC protocol; on the other hand, previous experience indicated that the compounds most likely to account for the antioxidant activity (i.e., carotenoids) are quantitatively extracted irrespective of whether ethanol/water (1:1, v/v) or acetone/hexane (1:1, v/v) are employed.
3.11. Mutagenicity Assessment
An Ames reversion assay was carried out, in duplicate, on the M2-1 extract (25, 50, 100 and 150 μL/plate) using two types of reference strains, with and without the S9 (liver microssomal fraction) mixture [15
]. Cell-free microalga extracts were accordingly prepared as described for the bacteriophage protection assay. The tester bacteria, Salmonella
Typhimurium strains TA 98 and TA 100, were characterized elsewhere [15
]. Reversion of specificity and activity of the S9 mix was confirmed with 5 μg of benzo-[a
]-pyrene per plate. Quercetin (1 μg/plate) was used as positive control, and plain solvent as negative one.