Spectrochimica Acta Part B 74–75 (2012) 169–176

Contents lists available at SciVerse ScienceDirect

Spectrochimica Acta Part B

j ourna l homepage: www.e lsev ie r .com/ locate /sab

Application of laser-induced breakdown spectroscopy to the analysis of algal biomassfor industrial biotechnology

P. Pořízka a, D. Prochazka a, Z. Pilát b, L. Krajcarová c, J. Kaiser a,⁎, R. Malina a, J. Novotný a, P. Zemánek b,J. Ježek b, M. Šerý b, S. Bernatová b, V. Krzyžánek b, K. Dobranská b, K. Novotný c, M. Trtílek d, O. Samek b

a X-ray micro CT and nano CT research group, CEITEC—Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, 616 00 Brno, Czech Republicb Institute of Scientific Instruments of the ASCR v.v.i., Academy of Sciences of the Czech Republic, Královopolská 147, Brno 61669, Czech Republicc Department of Chemistry, Faculty of Sciences, Masaryk University, Kotlářská 2, Brno 611 37, Czech Republicd Photon Systems Instruments, Drásov 470, 664 24 Drásov, Czech Republic

⁎ Corresponding author.E-mail address: kaiser@fme.vutbr.cz (J. Kaiser).

0584-8547/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.sab.2012.06.014

a b s t r a c t

a r t i c l e i n f o

Article history:Received 15 December 2011Accepted 18 June 2012Available online 26 June 2012

Keywords:LIBSDouble-pulseWater-jetAlgal biomassBiotechnology

We report on the application of laser-induced breakdown spectroscopy (LIBS) to the determination of ele-ments distinctive in terms of their biological significance (such as potassium, magnesium, calcium, and sodi-um) and to the monitoring of accumulation of potentially toxic heavy metal ions in living microorganisms(algae), in order to trace e.g. the influence of environmental exposure and other cultivation and biologicalfactors having an impact on them. Algae cells were suspended in liquid media or presented in a form of ad-herent cell mass on a surface (biofilm) and, consequently, characterized using their spectra. In our feasibilitystudy we used three different experimental arrangements employing double-pulse LIBS technique in order toimprove on analytical selectivity and sensitivity for potential industrial biotechnology applications, e.g. formonitoring of mass production of commercial biofuels, utilization in the food industry and control of the re-moval of heavy metal ions from industrial waste waters.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

One of the most promising alternatives to satisfy the increasingdemands of the human population for energy sources is the produc-tion of carbon-based fuels from plants. By the process of photosyn-thesis plants convert the energy of solar radiation into the chemicalenergy stored in the molecular building blocks of life (proteins, lipids,carbohydrates, etc.), thus providing energy for most of the life formson Earth. Moreover, photosynthesis has a crucial impact on Earth's at-mosphere as it generates oxygen while simultaneously using up car-bon dioxide, a prominent greenhouse-effect gas responsible for globalclimate changes.

In order to utilize photoautotrophic microorganisms (algae) for ef-ficient biofuel, food industry, and bioremediation applications [1,2]the optimal cultivation parameters have to be determined for eachgiven purpose, which results in a high production of oil in the selectedcell line, increased production of carotenoids/omega-3 oils and poten-tial absorption of heavy-metals in the selected cell line, respectively.This can be accomplished using small-scale photobioreactors thatallow precise monitoring and control of the culture irradiance, tem-perature, pH, and gas composition in the medium. However, the abil-ity to monitor the elemental composition of cells and consequently

rights reserved.

the cellular response to external stimuli in real time (ratios/elementalcompositions of algal cells might be significantly changed over shortperiods of time) is not provided. For this purpose qualitative/quantita-tive information about elemental composition of the algal cells thatprovides a deeper insight into the cellular physiology and enablesmore efficient optimization of the selected parameters for specifiedpurposes outlined above is required. So far, only a few studies [3–6]have been performed – using dedicated chemical techniques forlipid determination – to analyze the effect of nutrient elements onoil production/bioremediation. Thus, a fast and remote techniquewhich is capable to analyze elements in-situ within algal cells wouldbe of an advantage.

The technique of laser-induced breakdown spectroscopy (LIBS)utilizes the high power densities employing focused radiation froma pulsed, fixed-frequency laser in order to generate luminous plasmafrom a sample (solid, liquid, and gaseous samples) [7]. In our experi-ments we assume the stoichiometric ablation so that the plasma com-position reflects the elemental composition of the ablated target.

Here we report on the application of a double-pulse LIBS tech-nique to the analysis of important elements including heavy metalions in algal cells. The technique of double-pulse LIBS [8] was intro-duced by K. Niemax and his co-workers from ISAS Dortmund in1991 [9], and nowadays it is a common technique used in manyLIBS research laboratories. LIBS has been previously applied to theanalysis of biological samples [7,10–12], namely to the determinationof Sr in algal pellets [13].

Fig. 1. a). Schematic diagram of LIBS system for ablation of biofilm. b) Schematic diagram of LIBS system employing laminar water jet setup.

Fig. 2. Schematic diagram of experimental setup for bulk liquid LIBS experiments in-volving the two lasers. Ablation laser I (from the top) at 266nm and re-heating laserpulse II (from the side) at 1064nm are shown.

170 P. Pořízka et al. / Spectrochimica Acta Part B 74–75 (2012) 169–176

The realization of LIBS apparatus employing a water jet forreal-time, in situ and remote analysis of trace elements in liquid sam-ples, which is potentially applicable to the analysis of pollutants inwater in harsh or difficult-to-reach environments and used in our ex-periments, has been previously described [14–20].

Algal cells used for our investigations were present in liquid sus-pension (as was mentioned above for the use within the photo-bioreactor) or in a form of biofilm. Algal biomass in naturalenvironment may act as a bio-indicator of pollution. If some algal spe-cies are selectively added into the polluted area, they might serve as abioremedation tool.

We have utilized the LIBS set up in the three following modifica-tions to follow different applications demands:

(i) algal cells were suspended in a given volume of the liquid of in-terest and analyzed using double-pulse LIBS employing a waterjet (wet procedure);

(ii) algal cells were suspended in a given volume of the liquid of in-terest and analyzed using double-pulse LIBS employing bulkliquid measurements (wet procedure);

(iii) algal biofilm was analyzed using double-pulse LIBS (dryprocedure).

Valuable information about elemental composition of the cellsbrought by the LIBS technique can potentially help to elucidate im-portant questions in algal biology (nutrition dynamics depending onthe cultivation conditions) and identify the algal strains which havethe potential for applications in metal-ion sorption (bioremediation),in food industry (source of omega-3 oils and proteins [21]), or in abiofuel industry. Here we would like to note, that bioremediationcan be beneficially combined with the production of biofuels [22].For our experiments we have selected the two algal species—Trachydiscus minutus (potential source of omega-3 fatty acids suchas EPA and DHA) [23] and Chlamydomonas sp. (potential candidatefor biofuel production) [24].

To the best of our knowledge these are the first LIBS measure-ments utilizing setup with the liquid jet so that algae samples could

be measured in-vivo, on-line and in real-time. Obviously, this is thearea (real-world application) where LIBS technique can excel becausethe measurements can be performed directly e.g. at the bioreactor orremotely in difficult-to-reach environments at contaminated sites.

2. Experimental

As was mentioned above, in our investigations we used the threedifferent experimental arrangements using double-pulse LIBS tech-nique. Firstly, for fast, remote and in situ analysis we focused thelaser beam onto the smooth vertical surface of a laminar jet streamof the liquid similar to our previously published arrangement [17],

171P. Pořízka et al. / Spectrochimica Acta Part B 74–75 (2012) 169–176

secondly, onto the water surface (bulk liquid), very much as whenroutinely investigating solid targets. Thirdly, we set up a typical ar-rangement found in the majority of LIBS laboratories where laser isfocused onto the surface of algal biofilm.

2.1. Preparation of algal samples for laser ablation experiments

The algal species were obtained from the Culture Collection of Au-totrophic Organisms (CCALA), Institute of Botany, Academy of Sci-ences of the Czech Republic. This collection maintains about 650strains of microalgae and cyanobacteria, a substantial portion ofwhich are microorganisms from extreme environments. Recently,great effort has been put to the screening of microalgal strains suit-able for biotechnological exploitation with respect to specific metab-olites and production characteristics.

T. minutus (Bourrelly) was cultivated in 300ml batch cultures atroom temperature (approx. 22°C), in a medium containing (inmgl−1) N 150, P2O5 50, K2O 300, MgO 30, SO3 75, B 0.3, Cu 0.1, Fe 0.7,Mn 0.4, Mo 0.04, and Zn 0.3, dissolved in 1:1 distilled and tapwater mixture. The incident PAR flux density estimate was be-tween 100 and 300μmolphotonsm−2s−1. The cultivation apparatusconsisted of 1000ml Pyrex conical flasks fitted with a cotton plug,which were occasionally shaken manually. Lighting was supplied withtwo standard cool white 40W fluorescent tubes. Chlamydomonas sp.was cultivated in 150ml Erlenmeyer flasks in BBM medium at roomtemperature in daylight at a laboratorywindowwith occasionalmanual

Fig. 3. a, b Details of LIBS spectra for the algae biofilm sample. (a) Batch 1 – Cu solution wasbiofilm was prepared with adding Cu to the batch—both Cu lines are visible indicating accu

mixing. The cells were harvested in the stationary phase and subjectedto the treatment with increased concentrations of some metal ions.

The culture aliquots were each supplied with solutions of CuSO4

10mgml−1 in 1:1000 (v/v) ratio, and the cultures were incubatedfor 24hours with occasional shaking. The sedimented cells werewashed with distilled water and concentrated by a brief centrifuga-tion. For biomass/biofilm formation the sediment was then trans-ferred with spatula on the surface of a glass cover slip. The residualwater was left to evaporate in the room conditions in a semi-closeddustproof container. Dried samples were subjected to the laser abla-tion. In order to produce algal suspension the sedimented cells wereplaced to a defined amount of liquid.

We would like to note that the question of biofilm definition hasbeen debated in many discussions. In our study we favor the simpledefinition from a recently published review [25]—“a biofilm is a thincoating comprised of living material.”

2.2. LIBS experimental setup utilizing water jet (algal suspensions) andbiofilm

Measurements of algal samples on two different setups for exper-iments with samples in solid state, as biofilms, and in a form of liquid,as the algal suspension, were performed at Brno University of Tech-nology. We have employed setup with orthogonal pulses for biofilmexperiments because this configuration enables better mapping ofthe sample [26,27] to setup with collinear pulses, which was usedfor water jet analysis.

not added to the batch—clearly only spectra of Mg and K are shown; (b) Batch 2 – algalmulation of Cu within algae cells.

Fig. 4. a–d Segments of LIBS spectra, recorded from biofilm sample used in analysis highlighting individual elements—(a) Cu, (b) Mg, (c) K and (d) Na.

172 P. Pořízka et al. / Spectrochimica Acta Part B 74–75 (2012) 169–176

2.2.1. Biofilm experimentsThe double-pulse LIBS (DP LIBS) measurements were carried out

in a perpendicular geometry (Fig. 1a); this setup was previously de-scribed by our group [26,27]. The ablation laser was directed normalto the surface sample by mirrors and focused onto the sample surfaceby a 30mm focal length glass triplet. The beam of the second laserwas directed parallel to the sample surface in the plasma using mir-rors and focused on the plasma by the lens of 40mm focal length.The setup involved the two Q-switched Nd:YAG lasers both operatingat 10Hz. The first laser LQ 529a (SOLAR, BY) which operated at thesecond harmonic (532nm) with the pulse width of about 10ns wasused as the ablation source. The second laser Brilliant B (Quantel,FR) operating at the first harmonic (1064nm) with pulse width ofabout 6ns was used for reheating the plasma. The energy of the abla-tion laser pulse was set on 12mJ per pulse and the energy of the sec-ond laser was 110mJ per pulse, the laser focal diameter was ~80μm.

The sample was mounted on a stage with precision movements(2μm resolution) inside the ablation chamber (Tescan, a.s. CZ). Theablation spot was targeted and controlled by a CCD camera placedoutside of the ablation chamber.

2.2.2. Algal suspensionSetup for measuring liquid samples can be easily attached to DP

LIBS setup described above. Liquid measurements were performedin home-made glass vessel instead of interaction chamber Fig. 1b.

Continuous and steady thin flow of liquid has been achieved forliquid LIBS measurements. For this we used peristaltic pump (PCD81, Kouřil, CZ) working at 100ml/min. Liquid sample was introducedto the nozzle (diameter of 0.6mm) via silicone tubes and mounted tothe XY positioning stage (ThorLabs, US).

Pulses of both lasers were led through the optical system(ThorLabs, US/Newport, UK) in the collinear geometry, employingharmonic separator (Eksma Optics, LT) reflecting 1064nm and

transmitting 532nm. The lens with 75mm focal length focused thelaser beams into the thin flow of liquid and, consequently, luminousmicro-plasma was created.

In both above mentioned cases the LIBS plasma radiation was col-lected with UV-NIR achromatic collimating mirror system, the CC52(ANDOR, UK) and transported via a fiber optic system (25μm in di-ameter) connected to a spectrometer in the Echelle configuration(ME5000, Mechelle, ANDOR, UK). As a detector an ICCD camera(iStar 734i, ANDOR, UK) was employed.

The time-resolved studies were performed by controlling follow-ing parameters—the gate width tW (time during which the spectraare integrated), the gate delay time td to reduce the effect of continu-um signal in collected spectra (time delay after second laser pulseafter which the spectra are acquired by the detector), and the delaybetween the two pulses Δt. The two lasers and ICCD camera weretriggered via a delay generator (DG535, Stanford Research System,US). The best signal to noise ratio was obtained for the timing of op-timized setup tW=16μs, td=1.5μs, and Δt=1.5μs.

The LIBS analysis was performed in the air at atmospheric pres-sure. For each spectral window – obtained from the biofilm or fromalgal suspension – 10 laser shots were averaged. The gain level onan ICCD camera was set on 80.

2.3. LIBS experimental setup utilizing bulk liquid arrangement(algal suspension)

The measurements of algal suspensions were performed at theDepartment of Chemistry, Masaryk University in Brno employingthe orthogonal double-pulse LIBS setup (Fig. 2). The modified com-mercially available laser ablation system (UP 266 MACRO, NewWave, US), operating at the 266nm with energy of 12mJ per pulse,was used as the ablation laser. The energy of the ablation laser wasset on 12mJ using dedicated software. For the second, re-heating

173P. Pořízka et al. / Spectrochimica Acta Part B 74–75 (2012) 169–176

laser pulse which propagated parallel to the sample surface a Nd:YAGlaser (Brilliant, Quantel, FR) at fundamental wavelength 1064nm(with energy of 100mJ per pulse) was used. The laser beam was fo-cused by the 120mm focal length lens to intersect the path of the firstlaser beam and, consequently, to create microplasma placed 0.5mmabove the sample surface. The double pulse setup was controlled withtwo delay generators (DG 645, Stanford Research Systems, US) inorder to provide the time synchronization of both lasers and the ICCDdetector.

Each spectrum from the bulk liquid arrangement was obtainedfrom single laser shot and with no gain set on the camera. As the op-timal inter-pulse delay 600ns was set according to the highestsignal-to-noise ratio observed. The plasma emission was focused bythe 80mm focal length glass lens into the 3m long optical fiber andtransported into the entrance slit of Czerny-Turner spectrometer(TRIAX 320, Jobin Yvon, FR; 2400g/mm; 50μm entrance slit)equipped with ICCD detector (Horiba, Jobin Yvon, FR). The gatedelay time after second laser pulse and gate width of the detectionwere as well optimized to maximal signal to noise ratio and wereset to 500ns and 10μs, respectively.

Fig. 5. a, b (a) Overall view on the biofilm sample clearly showing ablated craters during LIBS1×1cm); (b) an image of the crater obtained from the confocal laser scanning microscope100μm.

3. Results and discussion

In this study, two different algal species—T. minutus andChlamydomonas sp. were examined in terms of the elemental compo-sition. We have detected magnesium, calcium, potassium, sodium,and copper. The corresponding atomic emission lines utilized wereas follows: Mg lines at 279.5nm and 280.2nm, Ca lines at 393.3nmand 396.8nm, K lines at 766.5nm and 769.8nm, Na lines 588.9nmand 589.6nm and Cu lines at 324.7nm and 327.4nm.

3.1. Measurements on algal biofilm—metal-ion binding experiments

LIBS experiments were performed on algal biofilm in order to re-veal elemental composition of a biological sample. We targeted thedried biofilm (prepared according to the procedure describedabove), so that “real-world” LIBS applications can be followed. As anemerging issue, the mechanism of metal-ion binding to algae wasstudied by LIBS on the two algal samples—firstly “batch 1” was pre-pared following normal protocol and secondly “batch 2” was treatedwith specified amount of Cu (as described above). Examples of the

analysis (in the middle of the sample, where 15 craters are located on the area of aboutfor the crater parameters estimation—depth of about 70μm and the diameter of about

Fig. 6. a–f Segments of LIBS spectra, recorded from water jet and bulk liquid arrangements used in analysis highlighting individual elements—(a) Ca, (b) Cu, (c) K and (d) Mg forwater jet and (e) Cu, and (f) Mg for bulk liquid setup (spectral range was limited by using the Czerny–Turner spectrometer).

174 P. Pořízka et al. / Spectrochimica Acta Part B 74–75 (2012) 169–176

spectra acquired from “batch 1” and “batch 2” are shown in Fig. 3aand b, respectively. The measured data clearly show the copperbinding capacity of Chlamydomonas sp. The copper binding capacitywas also measured for T. minutus (results are not shown) and wasfound slightly lower when monitoring Mg/Cu ratios (Mg belongsto the alkaline earth metals and its natural average concentrationin algal biomass is about 9mg/kg; Cu belongs to algal microelementswith natural concentration of about 0.02mg/kg [28]). Moreover,from the differences in the ratios of Mg/K (K belongs to the alkalimetals and its natural average concentration is about 4mg/kg [28])estimated for both the batches we can speculate that during the in-cubation when Cu was added nutritional conditions within cellshave been changed, that could mean Mg was preferentiallyabsorbed in the cells. It could suggest that alkali metals and alkalineearth elements were exchanged with microelements ions from theadded solution containing Cu. However, parameters such as concen-tration of metal in solution, pH, temperature, cations, anions andmetabolic stage of the cells can affect above results. Also, a surface

effect image analysis would be very useful so that metal sorptioncan be thoroughly investigated. Such studies are currently underprogression in our laboratories, exploiting LIBS and different imag-ing approaches.

These preliminary finding suggests that the algal biomass underour study might be potentially suitable for the removal of heavymetal ions from e.g. polluted waters. This was, indeed, as presentedabove, confirmed by our experiments which determined the bindingof Cu metal-ions by the presence of increasing levels of LIBS signalfor Cu lines. However, systematic studies are required to investigatemechanism of biofilm sorption further using statistical andchemometrics software, in order to improve on our analytical mea-surements. Such studies are currently under development in ourlaboratories, exploiting a selection of LIBS, Raman spectroscopyand LA-ICP-MS approaches. Specifically, we aim to identify andquantify elements within biofilms using LA-ICP-MS technique forthe purpose of cross-validation and Raman spectroscopy for algaloil determination.

175P. Pořízka et al. / Spectrochimica Acta Part B 74–75 (2012) 169–176

Fig. 4 shows segments of LIBS spectra, recorded from biofilm sam-ple presenting the capability of LIBS technique to detect different ele-ments within the algal cells. In order to estimate the ablationefficiency and the size of the ablation crater in the algal biofilm theconfocal laser scanning microscope (Olympus, LEXT OLS3100, JP) im-ages were obtained. Fig. 5a shows the ablated biofilm with the detailof the craters which were drilled during LIBS measurements. The ab-lation crater detail is shown on Fig. 5b presenting the crated depth ofabout 70μm and the diameter of about 100μm (the total thickness ofbiofilm was estimated of about 150μm). This translates to an averageablation rate to about 7μm per pulse, considering that LIBS analysiswas provided using 10 laser shots.

3.2. Measurements on algal suspension using water jet setup and bulkliquid setup

Here, LIBS experiments were performed on liquid suspensions ofalgal species. In the water jet example, shown in Fig. 6, biologicallyrelevant elements (Ca, K, Mg) are clearly visible confirming the feasi-bility of the LIBS water jet setup for qualitative/semi-quantitativeanalysis of algal suspension in liquids (T. minutus). The spectraobtained from the experiments employing water jet setup suggestthe possibility to perform detailed semi-quantitative analysis, whichis now being investigated in our laboratories. Also, as describedabove for measurements on biofilms, experiments with algae treatedwith Cu were performed using the water jet. Fig. 6b displays two Culines corresponding to copper that was adsorbed by algae. Conse-quently, these results were confirmed utilizing the bulk liquid setup; spectra are shown in Fig. 6e–f, confirming that both arrangementsare capable to measure traces of Cu in algae suspended in medium.We would like to point out the differences in the LIBS spectraobtained from the liquid jet and from the biofilm samples—intenseH and O lines are visible for the liquid jet (Fig. 6a–d) in contrary tothe strong lines of Ca for biofilm (Fig. 4).

The successful analysis of the elemental spectral signatures for thetwo algal samples confirms that LIBS spectroscopy could be used forfast on-line characterization of algal species within the industrial en-vironment. Measurements of the selected ranges of interest (e.g. forestimation of Mg/Cu, Ca/Mg, Mg/K ratios for semi-quantitative analy-sis) can be acquired in a few seconds, depending on the selectedrange and instrumentation. Thus, LIBS might be capable to offer areal-time, in-situ recognition of algal species in the future. Conse-quently, these (preliminary) results warrant more extensive investi-gations with larger collections of algal strains to evaluate the powerof LIBS spectroscopy compared to other analytical methods. This re-search is now being conducted in our laboratories.

4. Conclusion

In present study we have demonstrated that it is possible to per-form LIBS analysis of macro- and micro-element concentrations inalgal samples using the water jet setup, bulk liquid arrangementand by investigations utilizing biofilms. Additionally, the differencesrecognized using LIBS technique in the spectra of two algal batches,where one batch was exposed to medium containing elevated levelsof copper have been shown.

From our feasibility experiments LIBS proves to be a useful tool fordetecting absorption of pollutant elements by algae samples, whichmight be in turn considered as potential candidates for bioremedia-tion. The present study was undertaken as a proof of principle only,and thus we only focused on selected results from the two algal spe-cies. The results of our experiments conducted for samples of algalsuspensions and algal biofilms confirm LIBS as a fast, real-time tech-nique for remote analysis.

As a natural development we plan to combine LIBS with a tech-nique capable to analyze algal lipids (e.g. Raman spectroscopy) in

order to study the effects introduced by nutrient starvation – whichcan be detected/monitored using LIBS – on the oil production in se-lected algal samples. For this, we continue in our efforts to prove an-alytical potential of combining LIBS with Raman spectroscopy.

We believe that LIBS technique will be of significant assistance toresearch groups currently being involved in, or intending to join,the quest of sustainable biofuel generation, algal food applications,and bioremediation.

Acknowledgments

The authors acknowledge the support for O. Samek by a MarieCurie Re-integration Grant (PERG 06-GA-2009-256526). This workalso received support from the Ministry of Education, Youth andSports of the Czech Republic together with the European Commission,the Czech Science Foundation, and the Ministry of Industry and Tradeof the Czech Republic (projects ALISI No. CZ.1.05/2.1.00/01.0017, GAP205/11/1687, and FR-TI1/433). The work was also supported bythe project “CEITEC—Central European Institute of Technology”(CZ.1.05/1.1.00/02.0068) from European Regional DevelopmentFund.

References

[1] I. Khozin-Goldberg, U. Iskandarov, Z. Cohen, LC-PUFA from photosynthetic micro-algae: occurrence, biosynthesis, and prospects in biotechnology, Appl. Microbiol.Biotechnol. 91 (2011) 905–915.

[2] P. Jinfen, L. Ronggen, M. Li, A review of heavy metal adsorption by marine algae,Chin. J. Oceanol. Limnol. 18 (2000) 260–264.

[3] X. Deng, X. Fei, Y. Li, The effects of nutritional restriction on neutral lipid accu-mulation in Chlamydomonas and Chlorella, Afr. J. Microbiol. Res. 5 (2011)260–270.

[4] B.N. Tripathi, S.K. Metha, A. Amar, J.P. Gaur, Oxidative stress in Scenedesmus sp.during short‐ and long-term exposure to Cu2+ and Zn2+, Chemosphere 62(2006) 538–544.

[5] H.V. Perales-Vela, J.M. Pena-Castro, R.O. Canizares-Villanueva, Heavy metal de-toxification in eukaryotic microalgae, Chemosphere 64 (2006) 1–10.

[6] P.A. Terry, W. Stone, Biosorption of cadmium and copper contaminated water byScenedesmus abundans, Chemosphere 47 (2002) 249–255.

[7] A.W. Miziolek, V. Palleschi, I. Schechter, Laser-Induced Breakdown Spectroscopy(LIBS) Fundamentals and Applications, Cambridge University Press, Cambridge,2006..

[8] V.I. Babushok, F.C. DeLucia Jr., J.L. Gottfried, C.A. Munson, A.W. Miziolek, Doublepulse laser ablation and plasmas: laser induced breakdown spectroscopy signalenhancement, Spectrochim. Acta Part B 61 (2006) 999–1014.

[9] J. Uebbing, J. Brust, W. Sdorra, F. Leis, K. Niemax, Reheating of a laser producedplasma by a second pulse laser, Appl. Spectrosc. 45 (1991) 1419–1423.

[10] S.J. Rehse, J. Diedrich, S. Palchaudhuri, Identification and discrimination of Pseudo-monas aeruginosa bacteria grown in blood and bile by laser-induced breakdownspectroscopy, Spectrochim. Acta Part B 62 (2007) 1169–1176.

[11] J. Kaiser, M. Galiova, K. Novotny, R. Cervenka, L. Reale, J. Novotny, M. Liska, O.Samek, V. Kanicky, A. Hrdlicka, K. Stejskal, V. Adam, R. Kizek, Mapping of lead,magnesium and copper accumulation in plant tissues by laser-induced break-down spectroscopy and laser-ablation inductively coupled plasma mass spec-trometry, Spectrochim. Acta Part B 64 (2009) 67–73.

[12] K. Novotny, J. Kaiser, M. Galiova, V. Konecna, J. Novotny, R. Malina, M. Liska, V.Kanicky, V. Otruba, Mapping of different structures on large area of granitesample using laser-ablation based analytical techniques, an exploratory study,Spectrochim. Acta Part B 63 (2008) 1139–1144.

[13] L. Niu, H.H. Cho, K.S. Song, H.K. Cha, Y. Kim, Y.I. Lee, Direct determination of stron-tium in marine algal samples by laser-induced breakdown spectrometry, Appl.Spectrosc. 56 (2002) 1511–1514.

[14] Y. Feng, J. Yang, J. Fan, G. Yao, X. Ji, X. Zhang, X. Zheng, Z. Cui, Investigation oflaser-induced breakdown spectroscopy of a liquid jet, Appl. Opt. 49 (2010)70–74.

[15] M. Sadegh Cheri, S.H. Tavassoli, Quantitative analysis of toxic metals lead andcadmium in water jet by laser-induced breakdown spectroscopy, Appl. Opt. 50(2011) 1227–1233.

[16] P. Yaroshchyk, R.J.S. Morrison, D. Body, B.L. Cahdwick, Quantitative determinationof wear metals in engine oils using laser-induced breakdown spectroscopy: acomparison between liquid jets and static liquids, Spectrochim. Acta Part B 60(2005) 986–992.

[17] O. Samek, D.C.S. Beddows, J. Kaiser, S.V. Kukhlevsky, M. Liška, H.H. Telle, J. Young,Application of laser-induced breakdown spectroscopy to in-situ analysis of liquidsamples, Opt. Eng. 39 (2000) 2248–2262.

[18] G. Arca, A. Ciucci, V. Palleschi, S. Rastelli, E. Tognoni, Trace element analysis inwater by the laser-induced breakdown spectroscopy technique, Appl. Spectrosc.50 (1997) 1102–1105.

176 P. Pořízka et al. / Spectrochimica Acta Part B 74–75 (2012) 169–176

[19] S.D. Brown, M. Martin, S. Deshpande, S. Seal, K. Huang, E. Alm, Y. Yang, L. Wu, T.Yan, X. Liu, A. Arkin, K. Chourey, J. Zhou, D.K. Thompson, Cellular response ofShewanella oneidensis to strontium-stress, Appl. Environ. Microbiol. 72 (2006)890–900.

[20] N.K. Rai, A.K. Rai, LIBS—an efficient approach for the determination of Cr in indus-trial wastewater, J. Hazard. Mater. 150 (2008) 835–838.

[21] B.A. Rasala, M. Muto, J. Sullivan, S.P. Mayfield, Improved heterologousprotein expression in the chloroplast of Chlamydomonas reinhardtii throughpromoter and 5 untranslated region optimization, Plant Biotechnol. J. 9(2011) 674–683.

[22] J.B.K. Park, R.J. Craggs, A.N. Shilton, Wastewater treatment high rate algal pondsfor biofuel production, Bioresour. Technol. 102 (2011) 35–42.

[23] T. Řezanka, M. Petránková, V. Cepák, P. Přibyl, K. Singler, T. Cajthaml, Trachydiscusminutus, a new biotechnological source of eicosapentaenoic acid, Folie Microbiol.55 (2010) 265–269.

[24] O. Samek, A. Jonáš, Z. Pilát, P. Zemánek, L. Nedbal, J. Tříska, P. Kotas, M. Trtílek,Raman microspectroscopy of individual algal cells: sensing unsaturation of stor-age lipids in vivo, Sensors 10 (2010) 8635–8651.

[25] E. Kataran, P. Watnick, Signals, regulatory networks, and materials that build andbreak bacterial biofilms, Microbiol. Mol. Biol. Rev. 73 (2009) 310–347.

[26] M. Galiova, J. Kaiser, K. Novotny, M. Hartl, R. Kizek, P. Babula, Utilization oflaser-assisted analytical methods for monitoring of lead and nutrition elements dis-tribution in fresh and dried Capsicum annuum l. leaves, Microsc. Res. Tech. 74(2011) 845–852.

[27] M. Galiova, J. Kaiser, K. Novotny, M. Ivanov, M.N. Fisakova, L. Manicni, G. Tromba,T. Vaculovic, M. Liska, V. Kanicky, Investigation of the osteitis deformans phases insnake vertebrae by double-pulse laser-induced breakdown spectroscopy, Anal.Bioanal. Chem. 398 (2010) 1095–1107.

[28] I. Michalak, K. Chojnacka, K. Marycz, Using ICP-OES and SEM-EDX in biosorptionstudies, Microchim. Acta 172 (2011) 65–74.