Algal Biomass Analysis by Laser-Based Analytical Techniques—A Review
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paper_title: High-resolution analysis of trace elements in crustose coralline algae from the North Atlantic and North Pacific by laser ablation ICP-MS

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We have investigated the trace elemental composition in the skeleta of two specimens of attached-living ::: coralline algae of the species Clathromorphum compactum from the North Atlantic (Newfoundland) and ::: Clathromorphum nereostratum from the North Pacific/Bering Sea region (Amchitka Island, Aleutians). ::: Samples were analyzed using Laser Ablation-Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) ::: yielding for the first time continuous individual trace elemental records of up to 69 years in length. The ::: resulting algal Mg/Ca, Sr/Ca, U/Ca, and Ba/Ca ratios are reproducible within individual sample specimens. ::: Algal Mg/Ca ratios were additionally validated by electron microprobe analyses (Amchitka sample). Algal Sr/ ::: Ca, U/Ca, and Ba/Ca ratios were compared to algal Mg/Ca ratios, which previously have been shown to ::: reliably record sea surface temperature (SST). Ratios of Sr/Ca from both Clathromorphum species show a ::: strong positive correlation to temperature-dependent Mg/Ca ratios, implying that seawater temperature ::: plays an important role in the incorporation of Sr into algal calcite. Linear Sr/Ca-SST regressions have ::: provided positive, but weaker relationships as compared to Mg/Ca-SST relationships. Both, algal Mg/Ca and ::: Sr/Ca display clear seasonal cycles. Inverse correlations were found between algal Mg/Ca and U/Ca, Ba/Ca, ::: and correlations to SST are weaker than between Mg/Ca, Sr/Ca and SST. This suggests that the incorporation ::: of U and Ba is influenced by other factors aside from temperature
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paper_title: Production and harvesting of microalgae for wastewater treatment, biofuels, and bioproducts.

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The integration of microalgae-based biofuel and bioproducts production with wastewater treatment has major advantages for both industries. However, major challenges to the implementation of an integrated system include the large-scale production of algae and the harvesting of microalgae in a way that allows for downstream processing to produce biofuels and other bioproducts of value. Although the majority of algal production systems use suspended cultures in either open ponds or closed reactors, the use of attached cultures may offer several advantages. With regard to harvesting methods, better understanding and control of autoflocculation and bioflocculation could improve performance and reduce chemical addition requirements for conventional mechanical methods that include centrifugation, tangential filtration, gravity sedimentation, and dissolved air flotation. There are many approaches currently used by companies and industries using clean water at laboratory, bench, and pilot scale; however, large-scale systems for controlled algae production and/or harvesting for wastewater treatment and subsequent processing for bioproducts are lacking. Further investigation and development of large-scale production and harvesting methods for biofuels and bioproducts are necessary, particularly with less studied but promising approaches such as those involving attached algal biofilm cultures.
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paper_title: Triacylglycerol profiling of marine microalgae by mass spectrometry.

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We present a method for the determination of triacylglycerol (TAG) profiles of oleaginous saltwater microalgae relevant for the production of biofuels, bioactive lipids, and high-value lipid-based chemical precursors. We describe a technique to remove chlorophyll using quick, simple solid phase extraction (SPE) and directly compare the intact TAG composition of four microalgae species (Phaeodactylum tricornutum, Nannochloropsis salina, Nannochloropsis oculata, and Tetraselmis suecica) using MALDI time-of-flight (TOF) mass spectrometry (MS), ESI linear ion trap-orbitrap (LTQ Orbitrap) MS, and ¹H NMR spectroscopy. Direct MS analysis is particularly effective to compare the polyunsaturated fatty acid (PUFA) composition for triacylglycerols because oxidation can often degrade samples upon derivatization. Using these methods, we observed that T. suecica contains significant PUFA levels with respect to other microalgae. This method is applicable for high-throughput MS screening of microalgae TAG profiles and may aid in the commercial development of biofuels.
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paper_title: Freshening of the Alaska Coastal Current recorded by coralline algal Ba/Ca ratios

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Arctic Ocean freshening can exert a controlling influence on global climate, triggering ::: strong feedbacks on ocean‐atmospheric processes and affecting the global cycling of the ::: world’s oceans. Glacier‐fed ocean currents such as the Alaska Coastal Current are ::: important sources of freshwater for the Bering Sea shelf, and may also influence the Arctic ::: Ocean freshwater budget. Instrumental data indicate a multiyear freshening episode of ::: the Alaska Coastal Current in the early 21st century. It is uncertain whether this freshening ::: is part of natural multidecadal climate variability or a unique feature of anthropogenically ::: induced warming. In order to answer this, a better understanding of past variations in ::: the Alaska Coastal Current is needed. However, continuous long‐term high‐resolution ::: observations of the Alaska Coastal Current have only been available for the last 2 decades. ::: In this study, specimens of the long‐lived crustose coralline alga Clathromorphum ::: nereostratum were collected within the pathway of the Alaska Coastal Current and utilized ::: as archives of past temperature and salinity. Results indicate that coralline algal Mg/Ca ::: ratios provide a 60 year record of sea surface temperatures and track changes of the Pacific ::: Decadal Oscillation, a pattern of decadal‐to‐multidecadal ocean‐atmosphere climate ::: variability centered over the North Pacific. Algal Ba/Ca ratios (used as indicators of coastal ::: freshwater runoff) are inversely correlated to instrumentally measured Alaska Coastal ::: Current salinity and record the period of freshening from 2001 to 2006. Similar multiyear ::: freshening events are not evident in the earlier portion of the 60 year Ba/Ca record. ::: This suggests that the 21st century freshening of the Alaska Coastal Current is a unique ::: feature related to increasing glacial melt and precipitation on mainland Alaska.
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paper_title: Application of laser-induced breakdown spectroscopy to the analysis of algal biomass for industrial biotechnology

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Abstract We report on the application of laser-induced breakdown spectroscopy (LIBS) to the determination of elements distinctive in terms of their biological significance (such as potassium, magnesium, calcium, and sodium) 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 biological factors having an impact on them. Algae cells were suspended in liquid media or presented in a form of adherent cell mass on a surface (biofilm) and, consequently, characterized using their spectra. In our feasibility study we used three different experimental arrangements employing double-pulse LIBS technique in order to improve on analytical selectivity and sensitivity for potential industrial biotechnology applications, e.g. for monitoring of mass production of commercial biofuels, utilization in the food industry and control of the removal of heavy metal ions from industrial waste waters.
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paper_title: MALDI-typing of infectious algae of the genus Prototheca using SOM portraits.

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BACKGROUND ::: MALDI-typing has become a frequently used approach for the identification of microorganisms and recently also of invertebrates. Similarity-comparisons are usually based on single-spectral data. We apply self-organizing maps (SOM) to portray the MS-spectral data with individual resolution and to improve the typing of Prototheca algae by using meta-spectra representing prototypes of groups of similar-behaving single spectra. ::: ::: ::: RESULTS ::: The MALDI-TOF peaklists of more than 300 algae extracts referring to five Prototheca species were transformed into colored mosaic images serving as molecular portraits of the individual samples. The portraits visualize the algae-specific distribution of high- and low-amplitude peaks in two dimensions. Species-specific pattern of MS intensities were readily discernable in terms of unique single spots of high amplitude MS-peaks which collect characteristic fingerprint spectra. The spot patterns allow the visual identification of groups of samples referring to different species, genotypes or isolates. The use of meta-peaks instead of single-peaks reduces the dimension of the data and leads to an increased discriminating power in downstream analysis. ::: ::: ::: CONCLUSIONS ::: We expect that our SOM portray method improves MS-based classifications and feature selection in upcoming applications of MALDI-typing based species identifications especially of closely related species.
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paper_title: Biofuels from algae: challenges and potential.

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Algae biofuels may provide a viable alternative to fossil fuels; however, this technology must overcome a number of hurdles before it can compete in the fuel market and be broadly deployed. These challenges include strain identification and improvement, both in terms of oil productivity and crop protection, nutrient and resource allocation and use, and the production of co-products to improve the economics of the entire system. Although there is much excitement about the potential of algae biofuels, much work is still required in the field. In this article, we attempt to elucidate the major challenges to economic algal biofuels at scale, and improve the focus of the scientific community to address these challenges and move algal biofuels from promise to reality.
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paper_title: Establishment of a bioenergy-focused microalgal culture collection

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Abstract A promising renewable energy scenario involves growing photosynthetic microalgae as a biofuel feedstock that can be converted into fungible, energy-dense fuels. Microalgae transform the energy in sunlight into a variety of reduced-carbon storage products, including triacylglycerols, which can be readily transformed into diesel fuel surrogates. To develop an economically viable algal biofuel industry, it is important to maximize the production and accumulation of these targeted bioenergy carriers in selected strains. In an effort to identify promising feedstock isolates we developed, evaluated and optimized contemporary high-throughput cell-sorting techniques to establish a collection of microalgae isolated from highly diverse ecosystems near geographic areas that are potential sites for large-scale algal cultivation in the Southwest United States. These efforts resulted in a culture collection containing 360 distinct microalgal strains. We report on the establishment of this collection and some preliminary qualitative screening studies to identify important biofuel phenotypes including neutral lipid accumulation and growth rates. As part of this undertaking we determined suitable cultivation media and evaluated cryopreservation techniques critical for the long-term storage of the microorganisms in this collection. This technique allows for the rapid isolation of extensive strain biodiversity that can be leveraged for the selection of promising bioenergy feedstock strains, as well as for providing fundamental advances in our understanding of fundamental algal biology.
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paper_title: Laser-Induced Breakdown Spectroscopy (LIBS), Part II: Review of Instrumental and Methodological Approaches to Material Analysis and Applications to Different Fields

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The first part of this two-part review focused on the fundamental and diagnostics aspects of laser-induced plasmas, only touching briefly upon concepts such as sensitivity and detection limits and largely omitting any discussion of the vast panorama of the practical applications of the technique. Clearly a true LIBS community has emerged, which promises to quicken the pace of LIBS developments, applications, and implementations. With this second part, a more applied flavor is taken, and its intended goal is summarizing the current state-of-the-art of analytical LIBS, providing a contemporary snapshot of LIBS applications, and highlighting new directions in laser-induced breakdown spectroscopy, such as novel approaches, instrumental developments, and advanced use of chemometric tools. More specifically, we discuss instrumental and analytical approaches (e.g., double- and multi-pulse LIBS to improve the sensitivity), calibration-free approaches, hyphenated approaches in which techniques such as Raman and fluorescence are coupled with LIBS to increase sensitivity and information power, resonantly enhanced LIBS approaches, signal processing and optimization (e.g., signal-to-noise analysis), and finally applications. An attempt is made to provide an updated view of the role played by LIBS in the various fields, with emphasis on applications considered to be unique. We finally try to assess where LIBS is going as an analytical field, where in our opinion it should go, and what should still be done for consolidating the technique as a mature method of chemical analysis.
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paper_title: Determination of the total unsaturation in oils and margarines by fourier transform raman spectroscopy

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An improved Raman spectroscopic procedure for the determination of the total unsaturation in oils and fats using Fourier Transform Raman (FT-Raman) spectroscopy is described. An important advantage of FT-Raman for these samples is that the spectra are fluorescence-free unlike dispersive Raman which often uses visible excitation. Samples can be analyzed without any pre-treatment thus eliminating the need for dissolution in toxic solvents. The short acquisition time of FT-Raman and the ease of application allowed for a rapid sample turnover.
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paper_title: Bioactive compounds from cyanobacteria and microalgae: an overview.

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Cyanobacteria (blue-green algae) are photosynthetic prokaryotes used as food by humans. They have also been recognized as an excellent source of vitamins and proteins and as such are found in health food stores throughout the world. They are also reported to be a source of fine chemicals, renewable fuel and bioactive compounds. This potential is being realized as data from research in the areas of the physiology and chemistry of these organisms are gathered and the knowledge of cyanobacterial genetics and genetic engineering increased. Their role as antiviral, anti-tumour, antibacterial, anti-HIV and a food additive have been well established. The production of cyanobacteria in artificial and natural environments has been fully exploited. In this review the use of cyanobacteria and microalgae, production processes and biosynthesis of pigments, colorants and certain bioactive compounds are discussed in detail. The genetic manipulation of cyanobacteria and microalgae to improve their quality are also described at length.
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paper_title: A review of the biochemistry of heavy metal biosorption by brown algae.

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The passive removal of toxic heavy metals such as Cd(2+), Cu(2+), Zn(2+), Pb(2+), Cr(3+), and Hg(2+) by inexpensive biomaterials, termed biosorption, requires that the substrate displays high metal uptake and selectivity, as well as suitable mechanical properties for applied remediation scenarios. In recent years, many low-cost sorbents have been investigated, but the brown algae have since proven to be the most effective and promising substrates. It is their basic biochemical constitution that is responsible for this enhanced performance among biomaterials. More specifically, it is the properties of cell wall constituents, such as alginate and fucoidan, which are chiefly responsible for heavy metal chelation. In this comprehensive review, the emphasis is on outlining the biochemical properties of the brown algae that set them apart from other algal biosorbents. A detailed description of the macromolecular conformation of the alginate biopolymer is offered in order to explain the heavy metal selectivity displayed by the brown algae. The role of cellular structure, storage polysaccharides, cell wall and extracellular polysaccharides is evaluated in terms of their potential for metal sequestration. Binding mechanisms are discussed, including the key functional groups involved and the ion-exchange process. Quantification of metal-biomass interactions is fundamental to the evaluation of potential implementation strategies, hence sorption isotherms, ion-exchange constants, as well as models used to characterize algal biosorption are reviewed. The sorption behavior (i.e., capacity, affinity) of brown algae with various heavy metals is summarized and their relative performance is evaluated.
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paper_title: Spectroscopic analysis using a hybrid LIBS-Raman system

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A novel setup, combining two spectroscopic techniques, laser induced breakdown spectroscopy (LIBS) and Raman spectroscopy in a hybrid unit, is described. The work presented herein is part of a broader project that aims to demonstrate the applicability of the hybrid LIBS-Raman unit as an analytical tool for the investigation of samples and objects of cultural heritage. The system utilizes a nanosecond pulsed Nd:YAG laser (532 nm) for both LIBS and Raman analysis. In the Raman mode, a low intensity beam from the laser probes the sample surface and the scattering signal is collected into a grating spectrograph coupled to an intensified charge-coupled device (ICCD) detector, which records the Raman spectrum. In the LIBS mode a single high intensity pulse from the laser irradiates the sample surface and the time- and spectrally-resolved emission from the resulting laser ablation plume yields the LIBS spectrum. The use of a non-gated CCD detector was found to produce similar quality data (in terms of S/N ratio and fluorescence background) in the Raman mode, while in the LIBS mode spectral features were clearly broader but did not prevent identification of prominent atomic emission lines. Several model pigment samples were examined and the data obtained show the ability of the hybrid unit to record both Raman and LIBS spectra from the same point on the sample, a clear advantage over the use of different analytical setups.
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paper_title: Optimization of outdoor cultivation in flat panel airlift reactors for lipid production by Chlorella vulgaris.

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Microalgae are discussed as a potential renewable feedstock for biofuel production. The production of highly concentrated algae biomass with a high fatty acid content, accompanied by high productivity with the use of natural sunlight is therefore of great interest. In the current study an outdoor pilot plant with five 30 L Flat Panel Airlift reactors (FPA) installed southwards were operated in 2011 in Stuttgart, Germany. The patented FPA reactor works on the basis of an airlift loop reactor and offers efficient intermixing for homogeneous light distribution. A lipid production process with the microalgae Chlorella vulgaris (SAG 211-12), under nitrogen and phosphorous deprivation, was established and evaluated in regard to the fatty acid content, fatty acid productivity and light yield. In the first set of experiments limitations caused by restricted CO₂ availability were excluded by enriching the media with NaOH. The higher alkalinity allows a higher CO₂ content of supplied air and leads to doubling of fatty acid productivity. The second set of experiments focused on how the ratio of light intensity to biomass concentration in the reactor impacts fatty acid content, productivity and light yield. The specific light availability was specified as mol photons on the reactor surface per gram biomass in the reactor. This is the first publication based on experimental data showing the quantitative correlation between specific light availability, fatty acid content and biomass light yield for a lipid production process under nutrient deprivation and outdoor conditions. High specific light availability leads to high fatty acid contents. Lower specific light availability increases fatty acid productivity and biomass light yield. An average fatty acid productivity of 0.39 g L⁻¹ day⁻¹ for a 12 days batch process with a final fatty acid content of 44.6% [w/w] was achieved. Light yield of 0.4 g mol photons⁻¹ was obtained for the first 6 days of cultivation.
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paper_title: High-resolution Mg/Ca ratios in a coralline red alga as a proxy for Bering Sea temperature variations from 1902 to 1967

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We present the first continuous, high-resolution record of Mg/Ca variations within an encrusting coralline red alga, Clathromorphum nereostratum, from Amchitka Island, Aleutian Islands. Mg/Ca ratios of individual growth increments were analyzed by measuring a singlepoint, electron-microprobe transect, yielding a resolution of ~15 samples/year and a 65-year record (1902–1967) of variations. Results show that Mg/Ca ratios in the high-Mg calcite algal framework display ::: pronounced annual cyclicity and archive late spring–late fall sea-surface temperatures (SST) corresponding to the main season of algal growth. Mg/Ca values correlate well to local SST, as well as to an air temperature record from the same region. High spatial correlation to large-scale SST variability in the subarctic North Pacific is observed, with patterns of strongest correlation following the direction of major oceanographic features that play a key role in the exchange of water masses between the North Pacific and the Bering Sea. Our data correlate well with a shorter Mg/Ca record from ability of the alga to reliably record regional environmental signals. In addition, Mg/Ca ratios relate well to a 29-year δ18O time series measured on the same sample, providing additional support for the use of Mg in coralline red algae as a paleotemperature proxy that, unlike algal-δ18O, is not influenced by salinity fluctuations. Moreover, electron microprobe–based analysis enables higher sampling resolution and faster analysis, thus providing a promising approach for future studies of longer C. nereostratum records and applications to other coralline species.
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paper_title: A combined laser-induced breakdown and Raman spectroscopy Echelle system for elemental and molecular microanalysis

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Abstract Raman and laser-induced breakdown spectroscopy is integrated into a single system for molecular and elemental microanalyses. Both analyses are performed on the same ~ 0.002 mm 2 sample spot allowing the assessment of sample heterogeneity on a micrometric scale through mapping and scanning. The core of the spectrometer system is a novel high resolution dual arm Echelle spectrograph utilized for both techniques. In contrast to scanning Raman spectroscopy systems, the Echelle–Raman spectrograph provides a high resolution spectrum in a broad spectral range of 200–6000 cm − 1 without moving the dispersive element. The system displays comparable or better sensitivity and spectral resolution in comparison to a state-of-the-art scanning Raman microscope and allows short analysis times for both Raman and laser induced breakdown spectroscopy. The laser-induced breakdown spectroscopy performance of the system is characterized by ppm detection limits, high spectral resolving power (15,000), and broad spectral range (290–945 nm). The capability of the system is demonstrated with the mapping of heterogeneous mineral samples and layer by layer analysis of pigments revealing the advantages of combining the techniques in a single unified set-up.
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paper_title: In vivo NMR studies of higher plants and algae

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Publisher Summary This chapter discusses in vivo nuclear magnetic resonance (NMR) studies of higher plants and algae. As the opportunity arose for biologists to apply the emerging techniques of NMR spectroscopy to systems of biological interest, it was perhaps inevitable that they would first use NMR to study the properties of water in cells and tissues. The advantages of studying the water non-invasively, in an unperturbed system, were apparently only partly offset by the problems of interpretation that arose from the heterogeneity of living systems and a considerable literature developed in this field. High-resolution multinuclear NMR spectroscopy permits the detection of certain ions and metabolites in vivo , as well as the tissue water, and thus increases the potential enormously for tackling biochemical and physiological problems non-invasively; while NMR imaging, although still relying on the detection of the water signal, provides a method for mapping the spatial distribution of the water in the sample. The potential importance of these techniques to biologists and physiologists meant that their interests and requirements began to be reflected in the design of NMR equipment and this accelerated the application of the new techniques to physiological problems. In vivo NMR studies have always emphasized the non-invasive character of the investigation and the chapter illustrates how this important property is exploited in studies of higher plants and algae.
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paper_title: Anti-HIV Activity of Extracts and Compounds from Marine Algae

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Abstract In recent years, elucidation of novel bioactive substances from different marine organisms is gaining importance rapidly not only from the research and publications but also from controlled clinical studies of natural product-derived substances. They offer important leads for the development of antiviral drugs against viral infections caused by human immunodeficiency virus type 1 (HIV-1). Regarding this issue, numerous anti-HIV-1 therapeutic agents from marine resources have been reported for their potential medicine/medical application as novel functional ingredients in anti-HIV therapy. In detail, marine macroalgae have attracted much of attention as a reliable source for potential anti-HIV compounds. Up to date, several types of compounds such as tannins, polysaccharides, lectins, and derivatives have been isolated, identified, and reported to possess significant anti-HIV-1 activity.
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paper_title: Enhancing biomass energy yield from pilot-scale high rate algal ponds with recycling.

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This paper investigates the effect of recycling on biomass energy yield in High Rate Algal Ponds (HRAPs). Two 8 m(3) pilot-scale HRAPs treating primary settled sewage were operated in parallel and monitored over a 2-year period. Volatile suspended solids were measured from both HRAPs and their gravity settlers to determine biomass productivity and harvest efficiency. The energy content of the biomass was also measured. Multiplying biomass productivity and harvest efficiency gives the 'harvestable biomass productivity' and multiplying this by the energy content defines the actual 'biomass energy yield'. In Year 1, algal recycling was implemented in one of the ponds (HRAPr) and improved harvestable biomass productivity by 58% compared with the control (HRAPc) without recycling (HRAPr: 9.2 g/m(2)/d; HRAPc: 5.8 g/m(2)/d). The energy content of the biomass grown in HRAPr, which was dominated by Pediastrun boryanum, was 25% higher than the control HRAPc which contained a mixed culture of 4-5 different algae (HRAPr: 21.5 kJ/g; HRAPc: 18.6 kJ/g). In Year 2, HRAPc was then seeded with the biomass harvested from the P. boryanum dominated HRAPr. This had the effect of shifting algal dominance from 89% Dictyosphaerium sp. (which is poorly-settleable) to over 90% P. boryanum in 5 months. Operation of this pond was then switched to recycling its own harvested biomass, which maintained P. boryanum dominance for the rest of Year 2. This result confirms, for the first time in the literature, that species control is possible for similarly sized co-occurring algal colonies in outdoor HRAP by algal recycling. With regard to the overall improvement in biomass energy yield, which is a critical parameter in the context of algal cultivation for biofuels, the combined improvements that recycling triggered in biomass productivity, harvest efficiency and energy content enhanced the harvested biomass energy yield by 66% (HRAPr: 195 kJ/m(2)/day; HRAPc: 118 kJ/m(2)/day).
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paper_title: Microalgae as a raw material for biofuels production

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Biofuels demand is unquestionable in order to reduce gaseous emissions (fossil CO(2), nitrogen and sulfur oxides) and their purported greenhouse, climatic changes and global warming effects, to face the frequent oil supply crises, as a way to help non-fossil fuel producer countries to reduce energy dependence, contributing to security of supply, promoting environmental sustainability and meeting the EU target of at least of 10% biofuels in the transport sector by 2020. Biodiesel is usually produced from oleaginous crops, such as rapeseed, soybean, sunflower and palm. However, the use of microalgae can be a suitable alternative feedstock for next generation biofuels because certain species contain high amounts of oil, which could be extracted, processed and refined into transportation fuels, using currently available technology; they have fast growth rate, permit the use of non-arable land and non-potable water, use far less water and do not displace food crops cultures; their production is not seasonal and they can be harvested daily. The screening of microalgae (Chlorella vulgaris, Spirulina maxima, Nannochloropsis sp., Neochloris oleabundans, Scenedesmus obliquus and Dunaliella tertiolecta) was done in order to choose the best one(s), in terms of quantity and quality as oil source for biofuel production. Neochloris oleabundans (fresh water microalga) and Nannochloropsis sp. (marine microalga) proved to be suitable as raw materials for biofuel production, due to their high oil content (29.0 and 28.7%, respectively). Both microalgae, when grown under nitrogen shortage, show a great increase (approximately 50%) in oil quantity. If the purpose is to produce biodiesel only from one species, Scenedesmus obliquus presents the most adequate fatty acid profile, namely in terms of linolenic and other polyunsaturated fatty acids. However, the microalgae Neochloris oleabundans, Nannochloropsis sp. and Dunaliella tertiolecta can also be used if associated with other microalgal oils and/or vegetable oils.
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paper_title: Constraints to commercialization of algal fuels.

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Production of algal crude oil has been achieved in various pilot scale facilities, but whether algal fuels can be produced in sufficient quantity to meaningfully displace petroleum fuels, has been largely overlooked. Limitations to commercialization of algal fuels need to be understood and addressed for any future commercialization. This review identifies the major constraints to commercialization of transport fuels from microalgae. Algae derived fuels are expensive compared to petroleum derived fuels, but this could change. Unfortunately, improved economics of production are not sufficient for an environmentally sustainable production, or its large scale feasibility. A low-cost point supply of concentrated carbon dioxide colocated with the other essential resources is necessary for producing algal fuels. An insufficiency of concentrated carbon dioxide is actually a major impediment to any substantial production of algal fuels. Sustainability of production requires the development of an ability to almost fully recycle the phosphorous and nitrogen nutrients that are necessary for algae culture. Development of a nitrogen biofixation ability to support production of algal fuels ought to be an important long term objective. At sufficiently large scale, a limited supply of freshwater will pose a significant limitation to production even if marine algae are used. Processes for recovering energy from the algal biomass left after the extraction of oil, are required for achieving a net positive energy balance in the algal fuel oil. The near term outlook for widespread use of algal fuels appears bleak, but fuels for niche applications such as in aviation may be likely in the medium term. Genetic and metabolic engineering of microalgae to boost production of fuel oil and ease its recovery, are essential for commercialization of algal fuels. Algae will need to be genetically modified for improved photosynthetic efficiency in the long term.
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paper_title: Optimization of light use efficiency for biofuel production in algae.

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A major challenge for next decades is development of competitive renewable energy sources, highly needed to compensate fossil fuels reserves and reduce greenhouse gas emissions. Among different possibilities, which are currently under investigation, there is the exploitation of unicellular algae for production of biofuels and biodiesel in particular. Some algae species have the ability of accumulating large amount of lipids within their cells which can be exploited as feedstock for the production of biodiesel. Strong research efforts are however still needed to fulfill this potential and optimize cultivation systems and biomass harvesting. Light provides the energy supporting algae growth and available radiation must be exploited with the highest possible efficiency to optimize productivity and make microalgae large scale cultivation energetically and economically sustainable. Investigation of the molecular bases influencing light use efficiency is thus seminal for the success of this biotechnology. In this work factors influencing light use efficiency in algal biomass production are reviewed, focusing on how algae genetic engineering and control of light environment within photobioreactors can improve the productivity of large scale cultivation systems.
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paper_title: Coralline algal growth-increment widths archive North Atlantic climate variability

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Over the past decade coralline algae have increasingly been used as archives of paleoclimate information. Encrusting coralline algae, which deposit annual growth increments in a high Mg-calcite skeleton, are amongst the longest-lived shallow marine organisms. In fact, a live-collected plant has recently been shown to have lived for at least 850 years based on radiometric dating. While a number of investigations have successfully used geochemical information of coralline algal skeletons to reconstruct sea surface temperatures, less attention has been paid to employ growth increment widths as a temperature proxy. Here we explore the relationship between growth and environmental parameters in Clathromorphum compactum collected in the subarctic Northwestern Atlantic. Results indicate that growth-increment widths of individual plants are poorly correlated with instrumental sea surface temperatures (SST). However, an averaged record of multiple growth increment-width time series from a regional network of C. compactum specimens up to 800 km apart reveals strong correlations with annual instrumental SST since 1950. Hence, similar to methods applied in dendrochronology, averaging of multiple sclerochronological records of coralline algae provides accurate climate information. A 115-year growth-increment width master chronology created from modern-collected and museum specimens is highly correlated to multidecadal variability seen in North Atlantic sea surface temperatures. Positive changes in algal growth anomalies record the well-documented regime shift and warming in the northwestern Atlantic during the 1990s. Large positive changes in algal growth anomalies were also present in the 1920s and 1930s, indicating that the impact of a concurrent large-scale regime shift throughout the North Atlantic was more strongly felt in the subarctic Northwestern Atlantic than previously thought, and may have even exceeded the 1990s event with respect to the magnitude of the warming.
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paper_title: Structural similarities of fucoidans from brown algae Silvetia babingtonii and Fucus evanescens, determined by tandem MALDI-TOF mass spectrometry.

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Rapid mass spectrometric investigation of oligosaccharides, obtained by autohydrolysis of fucoidans from brown algae Silvetia babingtonii and Fucus evanescens (Fucales, Phaeophyceae) has shown both similarities and differences in structural features/sulfation pattern of their fragments, obtained in the same conditions. Tandem MALDI-TOF MS of fucooligosaccharides with even DP (degree of polymerization) was close to that observed for fucoidan from F. evanescens. Slight differences in tandem mass spectra of fragments with odd DP indicated, probably, sulfation at C-3 (instead of C-2 in F. evanescens) of some (1→4)-linked α-L-Fucp residues and/or the presence of short blocks, built up of (1→3)-linked α-L-Fucp residues.
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paper_title: Lipid extracts from different algal species:1H and13C-NMR spectroscopic studies as a new tool to screen differences in the composition of fatty acids, sterols and carotenoids

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One- and two-dimensional1H- and13C-NMR spectra of lipid extracts fromUlva rigida, Gracilaria longa, Fucus virsoides andCodium tomentosum collected in the northern Adriatic Sea allowed screening of the content of fatty acid chains, carotenoids, free and acylated cholesterol and chlorophylls. The carotenoid-to-polyunsaturated fatty acid molar ratio was taken as a comparison parameter in samples ofUlva rigida collected in differentloci and seasons; the value was markedly higher in samples from the Lagoon of Venice than from marine coastal waters. The total cholesterol concentration was evaluated by1H-NMR spectroscopy and similar values were found for all species. Two-dimensional heterocorrelated NMR spectroscopy was shown to give characteristic fingerprints of the lipid extracts from algal samples as regards the content in chlorophylls, unsaturated fatty acids and carotenoids.
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paper_title: Engineering challenges in biodiesel production from microalgae.

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In recent years, the not too distant exhaustion of fossil fuels is becoming apparent. Apart from this, the combustion of fossil fuels leads to environmental concerns, the emission of greenhouse gases and issues with global warming and health problems. Production of biodiesel from microalgae may represent an attractive solution to the above mentioned problems, and can offer a renewable source of fuel with fewer pollutants. This review presents a compilation of engineering challenges related to microalgae as a source of biodiesel. Advantages and current limitations for biodiesel production are discussed; some aspects of algae cells biology, with emphasis on cell wall composition, as it represents a barrier for fatty acid extraction and lipid droplets are also presented. In addition, recent advances in the different stages of the manufacturing process are included, starting from the strain selection and finishing in the processing of fatty acids into biodiesel.
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paper_title: Wastewater treatment high rate algal ponds for biofuel production.

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Abstract While research and development of algal biofuels are currently receiving much interest and funding, they are still not commercially viable at today’s fossil fuel prices. However, a niche opportunity may exist where algae are grown as a by-product of high rate algal ponds (HRAPs) operated for wastewater treatment. In addition to significantly better economics, algal biofuel production from wastewater treatment HRAPs has a much smaller environmental footprint compared to commercial algal production HRAPs which consume freshwater and fertilisers. In this paper the critical parameters that limit algal cultivation, production and harvest are reviewed and practical options that may enhance the net harvestable algal production from wastewater treatment HRAPs including CO 2 addition, species control, control of grazers and parasites and bioflocculation are discussed.
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paper_title: Raman Microspectroscopy of Individual Algal Cells: Sensing Unsaturation of Storage Lipids in vivo

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Algae are becoming a strategic source of fuels, food, feedstocks, and biologically active compounds. This potential has stimulated the development of innovative analytical methods focused on these microorganisms. Algal lipids are among the most promising potential products for fuels as well as for nutrition. The crucial parameter characterizing the algal lipids is the degree of unsaturation of the constituent fatty acids quantified by the iodine value. Here we demonstrate the capacity of the spatially resolved Raman microspectroscopy to determine the effective iodine value in lipid storage bodies of individual living algal cells. The Raman spectra were collected from three selected algal species immobilized in an agarose gel. Prior to immobilization, the algae were cultivated in the stationary phase inducing an overproduction of lipids. We employed the characteristic peaks in the Raman scattering spectra at 1,656 cm−1 (cis C═C stretching mode) and 1,445 cm−1 (CH2 scissoring mode) as the markers defining the ratio of unsaturated-to-saturated carbon-carbon bonds of the fatty acids in the algal lipids. These spectral features were first quantified for pure fatty acids of known iodine value. The resultant calibration curve was then used to calculate the effective iodine value of storage lipids in the living algal cells from their Raman spectra. We demonstrated that the iodine value differs significantly for the three studied algal species. Our spectroscopic estimations of the iodine value were validated using GC-MS measurements and an excellent agreement was found for the Trachydiscus minutus species. A good agreement was also found with the earlier published data on Botryococcus braunii. Thus, we propose that Raman microspectroscopy can become technique of choice in the rapidly expanding field of algal biotechnology.
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paper_title: Use of LIDAR in landslide investigations: a review

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This paper presents a short history of the appraisal of laser scanner technologies in geosciences used for imaging relief by high-resolution digital elevation models (HRDEMs) or 3D models. A general overview of light detection and ranging (LIDAR) techniques applied to landslides is given, followed by a review of different applications of LIDAR for landslide, rockfall and debris-flow. These applications are classified as: (1) Detection and characterization of mass movements; (2) Hazard assessment and susceptibility mapping; (3) Modelling; (4) Monitoring. This review emphasizes how LIDAR-derived HRDEMs can be used to investigate any type of landslides. It is clear that such HRDEMs are not yet a common tool for landslides investigations, but this technique has opened new domains of applications that still have to be developed.
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paper_title: Coralline algal Barium as indicator for 20th century northwestern North Atlantic surface ocean freshwater variability

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During the past decades climate and freshwater dynamics in the northwestern North Atlantic have undergone major changes. Large-scale freshening episodes, related to polar freshwater pulses, have had a strong influence on ocean variability in this climatically important region. However, little is known about variability before 1950, mainly due to the lack of long-term high-resolution marine proxy archives. Here we present the first multidecadal-length records of annually resolved Ba/Ca variations from Northwest Atlantic coralline algae. We observe positive relationships between algal Ba/Ca ratios from two Newfoundland sites and salinity observations back to 1950. Both records capture episodical multi-year freshening events during the 20th century. Variability in algal Ba/Ca is sensitive to freshwater-induced changes in upper ocean stratification, which affect the transport of cold, Ba-enriched deep waters onto the shelf (highly stratified equals less Ba/Ca). Algal Ba/Ca ratios therefore may serve as a new resource for reconstructing past surface ocean freshwater changes.
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paper_title: Anticancer compounds from marine macroalgae and their application as medicinal foods.

paper_content:

Cancer is one of the most challenging medical conditions that need a proper therapeutic approach for its proper management with fewer side effects. Until now, many of the phytochemicals from terrestrial origin have been assessed for their anticancer ability and few of them are in clinical trials too. However, marine environment also has been a greatest resource that harbors taxonomically diverse and a variety of life forms and serves as store house for several biologically beneficial metabolites. Hitherto, many metabolites have been isolated from marine biomasses that have exhibited excellent biological activities, especially as anticancer agents. In particular, marine macroalgae which are considered as dietary constituents in Pacific Asian region have become chief resources for their unparalleled and unique metabolites like sulfated polysaccharides (SPs), phlorotannins, and their ability in reducing the risk of cancer and its related diseases. In this chapter, we have discussed the anticancer activities of marine algae-derived SPs, phlorotannins, and carotenoids and the possibilities of marine algae as potential medicinal foods in the management of cancer.
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paper_title: Use of algae as biofuel sources

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The aim of this study is to investigate the algae production technologies such as open, closed and hybrid systems, production costs, and algal energy conversions. Liquid biofuels are alternative fuels promoted with potential to reduce dependence on fossil fuel imports. Biofuels production costs can vary widely by feedstock, conversion process, scale of production and region. Algae will become the most important biofuel source in the near future. Microalgae appear to be the only source of renewable biodiesel that is capable of meeting the global demand for transport fuels. Microalgae can be converted to bio-oil, bioethanol, bio-hydrogen and bimethane via thermochemical and biochemical methods. Microalgae are theoretically very promising source of biodiesel.
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paper_title: Sustainability of algal biofuel production using integrated renewable energy park (IREP) and algal biorefinery approach

paper_content:

Algal biomass can provide viable third generation feedstock for liquid transportation fuel. However, for a mature commercial industry to develop, sustainability as well as technological and economic issues pertinent to algal biofuel sector must be addressed first. This viewpoint focuses on three integrated approaches laid out to meet these challenges. Firstly, an integrated algal biorefinery for sequential biomass processing for multiple high-value products is delineated to bring in the financial sustainability to the algal biofuel production units. Secondly, an integrated renewable energy park (IREP) approach is proposed for amalgamating various renewable energy industries established in different locations. This would aid in synergistic and efficient electricity and liquid biofuel production with zero net carbon emissions while obviating numerous sustainability issues such as productive usage of agricultural land, water, and fossil fuel usage. A 'renewable energy corridor' rich in multiple energy sources needed for algal biofuel production for deploying IREPs in the United States is also illustrated. Finally, the integration of various industries with algal biofuel sector can bring a multitude of sustainable deliverables to society, such as renewable supply of cheap protein supplements, health products and aquafeed ingredients. The benefits, challenges, and policy needs of the IREP approach are also discussed.
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paper_title: Optical Algal Biosensor using Alkaline Phosphatase for Determination of Heavy Metals

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A biosensor is constructed to detect heavy metals from inhibition of alkaline phosphatase (AP) present on the external membrane of Chlorella vulgaris microalgae. The microalgal cells are immobilized on removable membranes placed in front of the tip of an optical fiber bundle inside a homemade microcell. C. vulgaris was cultivated in the laboratory and its alkaline phosphatase activity is strongly inhibited in the presence of heavy metals. This property has been used for the determination of those toxic compounds.
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paper_title: Comparing several atomic spectrometric methods to the super stars: special emphasis on laser induced breakdown spectrometry, LIBS, a future super star

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The “super stars” of analytical atomic spectrometry are electrothermal atomization-atomic absorption spectrometry (ETA-AAS), inductively coupled plasma-atomic emission spectrometry (ICP-AES) and inductively coupled plasma-mass spectrometry (ICP-MS). Many other atomic spectrometric methods have been used to determine levels of elements present in solid, liquid and gaseous samples, but in most cases these other methods are inferior to the big three super star methods. The other atomic methods include glow discharge emission, absorption and mass spectrometric methods, laser excited fluorescence emission and ionization methods, and flame and microwave plasma emission and mass spectrometric methods. These “lesser” methods will be compared to the “super star” methods based on a number of figures of merit, including detection power, selectivity, multi-element capability, cost, applications, and “age” of the methods. The “age” of the method will be determined by a modification of the well-known Laitinen “Seven Ages of an Analytical Method” ::: (H.A. Laitinen, Anal. Chem., 1973, 45, 2305). Calculations will show that certain methods are capable of single atom detection, including several atomic absorption methods, as well as laser atomic ionization and fluorescence methods. The comparison of methods will indicate why the “super stars” of atomic spectrometric methods will continue to retain their status and what must be done for the lesser atomic methods to approach “super star” status. Certainly most of the lesser atomic spectrometric methods will have a limited place in the analytical arena. Because of the wide current interest and research activity, special emphasis will be placed on the technique of laser induced breakdown spectrometry (LIBS). Its current status and future developments will therefore be reviewed.
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paper_title: Potential for green microalgae to produce hydrogen, pharmaceuticals and other high value products in a combined process

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Green microalgae for several decades have been produced for commercial exploitation, with applications ranging from health food for human consumption, aquaculture and animal feed, to coloring agents, cosmetics and others. Several products from green algae which are used today consist of secondary metabolites that can be extracted from the algal biomass. The best known examples are the carotenoids astaxanthin and β-carotene, which are used as coloring agents and for health-promoting purposes. Many species of green algae are able to produce valuable metabolites for different uses; examples are antioxidants, several different carotenoids, polyunsaturated fatty acids, vitamins, anticancer and antiviral drugs. In many cases, these substances are secondary metabolites that are produced when the algae are exposed to stress conditions linked to nutrient deprivation, light intensity, temperature, salinity and pH. In other cases, the metabolites have been detected in algae grown under optimal conditions, and little is known about optimization of the production of each product, or the effects of stress conditions on their production. Some green algae have shown the ability to produce significant amounts of hydrogen gas during sulfur deprivation, a process which is currently studied extensively worldwide. At the moment, the majority of research in this field has focused on the model organism, Chlamydomonas reinhardtii, but other species of green algae also have this ability. Currently there is little information available regarding the possibility for producing hydrogen and other valuable metabolites in the same process. This study aims to explore which stress conditions are known to induce the production of different valuable products in comparison to stress reactions leading to hydrogen production. Wild type species of green microalgae with known ability to produce high amounts of certain valuable metabolites are listed and linked to species with ability to produce hydrogen during general anaerobic conditions, and during sulfur deprivation. Species used today for commercial purposes are also described. This information is analyzed in order to form a basis for selection of wild type species for a future multi-step process, where hydrogen production from solar energy is combined with the production of valuable metabolites and other commercial uses of the algal biomass.
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paper_title: Dual role of microalgae: Phycoremediation of domestic wastewater and biomass production for sustainable biofuels production

paper_content:

Global threats of fuel shortages in the near future and climate change due to green-house gas emissions are posing serious challenges and hence and it is imperative to explore means for sustainable ways of averting the consequences. The dual application of microalgae for phycoremediation and biomass production for sustainable biofuels production is a feasible option. The use of high rate algal ponds (HRAPs) for nutrient removal has been in existence for some decades though the technology has not been fully harnessed for wastewater treatment. Therefore this paper discusses current knowledge regarding wastewater treatment using HRAPs and microalgal biomass production techniques using wastewater streams. The biomass harvesting methods and lipid extraction protocols are discussed in detail. Finally the paper discusses biodiesel production via transesterification of the lipids and other biofuels such as biomethane and bioethanol which are described using the biorefinery approach.
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paper_title: Potential of Near-Infrared Fourier Transform Raman Spectroscopy in Food Analysis

paper_content:

The 1064-nm excited Fourier transform (FT) Raman spectra have been measured in situ for various foods in order to investigate the potential of near-infrared (NIR) FT-Raman spectroscopy in food analysis. It is demonstrated here that NIR FT-Raman spectroscopy is a very powerful technique for (1) detecting selectively the trace components in foodstuffs, (2) estimating the degree of unsaturation of fatty acids included in foods, (3) investigating the structure of food components, and (4) monitoring changes in the quality of foods. Carotenoids included in foods give two intense bands near 1530 and 1160 cm−1 via the pre-resonance Raman effect in the NIR FT-Raman spectra, and therefore, the NIR FT-Raman technique can be employed to detect them nondestructively. Foods consisting largely of lipids such as oils, tallow, and butter show bands near 1658 and 1443 cm−1 due to C=C stretching modes of cis unsaturated fatty acid parts and CH2 scissoring modes of saturated fatty acid parts, respectively. It has been found that there is a linear correlation for various kinds of lipid-containing foods between the iodine value (number) and the intensity ratio of two bands at 1658 and 1443 cm−1 (I1658/I1443), indicating that the ratio can be used as a practical indicator for estimating the unsaturation level of a wide range of lipid-containing foods. A comparison of the Raman spectra of raw and boiled egg white shows that the amide I band shifts from 1666 to 1677 cm−1 and the intensity of the amide III band at 1275 cm−1 decreases upon boiling. These observations indicate that most α-helix structure changes into unordered structure in the proteins constituting egg white upon boiling. The NIR FT-Raman spectrum of old-leaf (about one year old) Japanese tea has been compared with that of its new leaf. The intensity ratio of two bands at 1529 and 1446 cm−1 (I1579/I1446), assignable to carotenoid and proteins, respectively, is considerably smaller in the former than in the latter, indicating that the ratio is useful for monitoring the changes in the quality of Japanese tea.
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paper_title: Trace elemental analysis by laser-induced breakdown spectroscopy—Biological applications

paper_content:

Abstract Laser-Induced Breakdown Spectroscopy (LIBS) is a sensitive optical technique capable of fast multi-elemental analysis of solid, gaseous and liquid samples. Since the late 1980s LIBS became visible in the analytical atomic spectroscopy scene; its applications having been developed continuously since then. In this paper, the use of LIBS for trace element determination in different matrices is reviewed. The main emphasis is on spatially resolved analysis of microbiological, plant and animal samples.
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paper_title: Laser-induced breakdown spectroscopy (LIBS): an overview of recent progress and future potential for biomedical applications.

paper_content:

The recent progress made in developing laser-induced breakdown spectroscopy (LIBS) has transformed LIBS from an elemental analysis technique to one that can be applied for the reagentless analysis of molecularly complex biological materials or clinical specimens. Rapid advances in the LIBS technology have spawned a growing number of recently published articles in peer-reviewed journals which have consistently demonstrated the capability of LIBS to rapidly detect, biochemically characterize and analyse, and/or accurately identify various biological, biomedical or clinical samples. These analyses are inherently real-time, require no sample preparation, and offer high sensitivity and specificity. This overview of the biomedical applications of LIBS is meant to summarize the research that has been performed to date, as well as to suggest to health care providers several possible specific future applications which, if successfully implemented, would be significantly beneficial to humankind.
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paper_title: Application of laser-induced breakdown spectroscopy to the analysis of algal biomass for industrial biotechnology

paper_content:

Abstract We report on the application of laser-induced breakdown spectroscopy (LIBS) to the determination of elements distinctive in terms of their biological significance (such as potassium, magnesium, calcium, and sodium) 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 biological factors having an impact on them. Algae cells were suspended in liquid media or presented in a form of adherent cell mass on a surface (biofilm) and, consequently, characterized using their spectra. In our feasibility study we used three different experimental arrangements employing double-pulse LIBS technique in order to improve on analytical selectivity and sensitivity for potential industrial biotechnology applications, e.g. for monitoring of mass production of commercial biofuels, utilization in the food industry and control of the removal of heavy metal ions from industrial waste waters.
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paper_title: Handbook of Laser-Induced Breakdown Spectroscopy

paper_content:

Foreword. Preface. Acronyms, Constants, And Symbol.s 1. History. 1.1 Atomic optical emission spectrochemistry (OES). 1.2 Laser-induced breakdown spectroscopy (LIBS). 1.3 LIBS History 1960-1980. 1.4 LIBS History 1980-1990. 1.5 LIBS History 1990-2000. 1.6 Active Areas of Investigation, 2000-2002. References. 2. Basics of the LIBS plasma. 2.1 LIBS plasma fundamentals. 2.2 laser-Induced Breakdown. 2.3 laser ablation. 2.4 double or multiple pulse libs. 2.5 summary. References. 3. Apparatus fundamentals. 3.1 Basic LIBS apparatus. 3.2 Lasers. 3.3 Optical systems. 3.4 Methods of spectral resolution. 3.5 Detectors. 3.6 Detection system calibration. 3.7 Timing considerations. 3.8 Methods of LIBS deployment. References. 4. Determining LIBS analytical figures-of-merit. 4.1 Introduction. 4.2 Basics of LIBS measurements. 4.3 precision. 4.4 Calibration. 4.5 Detection limit. References. 5. Qualitative LIBS Analysis. 5.1 Identifying elements. 5.2 Material identification. 5.3 Process control. References. 6. Quantitative LIBS Analysis. 6.1 Introduction. 6.2 Geometric Sampling Parameters. 6.3 Other sampling considerations. 6.4. Particle size. 6.5 use of internal standardization. 6.6 Chemical Matrix effects. 6.7. Example of libs measurement: Impurities in Lithium Solutions. 6.8 Reported figures of merit for LIBS measurements. 6.9 Conclusions. References. Chapter 7. REMOTE LIBS MEASUREMENTS. 7.1 Introduction. 7.2 Conventional open path LIBS. 7.3 Stand-off LIBS using Femtosecond pulses. 7.4 Fiber optic LIBS. References 8. Examples of recent LIBS fundamental research, instruments and novel applications. 8.1 Introduction. 8.2 fundamentals. 8.3 calibration-free LIBS. 8.4 laser and spectrometer advances. 8.5 surface analysis. 8.6 Double pulse studies and applications. 8.7 Steel applications. 8.8 libs for biological applications. 8.9 nuclear reactor applications. 8.10 LIBS for space applications. References. 9. THE FUTURE OF LIBS. 9.1 Introduction. 9.2 Expanding the understanding and capability of the libs process. 9.3 Widening the universe of libs applications. 9.4 Factors that will speed the commercialization of Libs. 9.5 conclusion. References. APPENDIX A: Safety Considerations in LIBS. A.1. safety plans. A.2 Laser Safety. A.3 Generation of Aerosols. A.4 laser pulse induced ignition. APPENDIX B: LIBS Application Matrix. APPENDIX C: LIBS Detection Limits. C.1 detection limits from the literature. C.2 uniform detection limits. APPENDIX D: Major LIBS References. Index.
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paper_title: Laser-Induced Breakdown Spectroscopy (LIBS), Part I: Review of Basic Diagnostics and Plasma—Particle Interactions: Still-Challenging Issues within the Analytical Plasma Community

paper_content:

Laser-induced breakdown spectroscopy (LIBS) has become a very popular analytical method in the last decade in view of some of its unique features such as applicability to any type of sample, practically no sample preparation, remote sensing capability, and speed of analysis. The technique has a remarkably wide applicability in many fields, and the number of applications is still growing. From an analytical point of view, the quantitative aspects of LIBS may be considered its Achilles' heel, first due to the complex nature of the laser-sample interaction processes, which depend upon both the laser characteristics and the sample material properties, and second due to the plasma-particle interaction processes, which are space and time dependent. Together, these may cause undesirable matrix effects. Ways of alleviating these problems rely upon the description of the plasma excitation-ionization processes through the use of classical equilibrium relations and therefore on the assumption that the laser-induced plasma is in local thermodynamic equilibrium (LTE). Even in this case, the transient nature of the plasma and its spatial inhomogeneity need to be considered and overcome in order to justify the theoretical assumptions made. This first article focuses on the basic diagnostics aspects and presents a review of the past and recent LIBS literature pertinent to this topic. Previous research on non-laser-based plasma literature, and the resulting knowledge, is also emphasized. The aim is, on one hand, to make the readers aware of such knowledge and on the other hand to trigger the interest of the LIBS community, as well as the larger analytical plasma community, in attempting some diagnostic approaches that have not yet been fully exploited in LIBS.
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paper_title: Laser-Induced Breakdown Spectroscopy: Fundamentals and Applications

paper_content:

Introduction.- Laser-induced breakdown spectroscopy.- Process parameters.- Instrumental components.- Evaporation and plasma generation.- Multiple-pulses for LIBS.- Material ablation.- Plasma dynamics and plasma parameters.- Plasma emission.- Modeling of plasma emission.- Quantitative analysis.- Combination of LIBS and LIF.- Bulk analysis of metallic alloys.- Bulk analysis of non-conducting materials.- Spatially resolved analysis.- Depth profiling.- LIBS instruments.- Industrial applications.
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paper_title: Laser-Induced Breakdown Spectroscopy (LIBS), Part II: Review of Instrumental and Methodological Approaches to Material Analysis and Applications to Different Fields

paper_content:

The first part of this two-part review focused on the fundamental and diagnostics aspects of laser-induced plasmas, only touching briefly upon concepts such as sensitivity and detection limits and largely omitting any discussion of the vast panorama of the practical applications of the technique. Clearly a true LIBS community has emerged, which promises to quicken the pace of LIBS developments, applications, and implementations. With this second part, a more applied flavor is taken, and its intended goal is summarizing the current state-of-the-art of analytical LIBS, providing a contemporary snapshot of LIBS applications, and highlighting new directions in laser-induced breakdown spectroscopy, such as novel approaches, instrumental developments, and advanced use of chemometric tools. More specifically, we discuss instrumental and analytical approaches (e.g., double- and multi-pulse LIBS to improve the sensitivity), calibration-free approaches, hyphenated approaches in which techniques such as Raman and fluorescence are coupled with LIBS to increase sensitivity and information power, resonantly enhanced LIBS approaches, signal processing and optimization (e.g., signal-to-noise analysis), and finally applications. An attempt is made to provide an updated view of the role played by LIBS in the various fields, with emphasis on applications considered to be unique. We finally try to assess where LIBS is going as an analytical field, where in our opinion it should go, and what should still be done for consolidating the technique as a mature method of chemical analysis.
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paper_title: The development of fieldable laser-induced breakdown spectrometer: No limits on the horizon

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In this review, new trends in the development of fieldable instrumentation based on laser-induced breakdown spectroscopy (LIBS) and its recent applications is presented. Depending on the LIBS configuration we will distinguish between portable, remote and stand-off instruments. Moreover, the development of portable systems gives greater flexibility and also increases the range of LIBS applications. In general, portable instruments are employed in close-contact applications like immovable artworks, contaminated soils and environmental diagnostic, while remote and stand-off instruments are normally used in analytical applications at distances where access to the sample is difficult or hazardous. Although remote and stand-off instruments are both used for chemical analysis at distances, the instrumental configurations are completely different. In remote analysis, an optical fiber is employed to deliver the laser energy a certain distance. This approach has been usually restricted to industrial applications, bulk analysis in water, geological measurements and chemical analysis on nuclear stations. In the case of stand-off applications, the laser beam and the returning plasma light are transmitted in an open-path configuration. In this article we also discuss the instrumental requirements in the design of remote and stand-off instruments.
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paper_title: Characterization of laser induced plasmas by optical emission spectroscopy: A review of experiments and methods

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Advances in characterization of laser induced plasmas by optical emission spectroscopy are reviewed in this article. The review is focused on the progress achieved in the determination of the physical parameters characteristic of the plasma, such as electron density, temperature and densities of atoms and ions. The experimental issues important for characterization by optical emission spectroscopy, as well as the different measurement methods are discussed. The main assumptions of the methods, namely the optical thin emission of spectral lines and the existence of local thermodynamic equilibrium in the plasma are evaluated. For dense and inhomogeneous sources of radiation such as laser induced plasmas, the characterization methods are classified in terms of the optical depth and the spatial resolution of the emission used for the measurements. The review deals firstly with optically thin spatially integrated measurements. Next, local measurements and characterization in not optically thin conditions are discussed. Two tables are included that provide reference to the works reporting measurements of electron density and temperature of laser induced plasmas generated with diverse samples.
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paper_title: Laser-induced breakdown spectroscopy (LIBS) : fundamenls and applications

paper_content:

Preface R. Russo and A. W. Miziolek 1. History and fundamentals of LIBS D. A. Cremers and L. J. Radziemski 2. Plasma morphology I. Schechter and V. Bulatov 3. From sample to signal in laser induced breakdown spectroscopy: a complex route to quantitative analysis E. Tognoni, V. Palleschi, M. Corsi, G. Cristoforetti, N. Omenetto, I. Gornushkin, B. W. Smith and J. D. Winefordner 4. Laser induced breakdown in gases: experiments and simulation C. G. Parigger 5. Analysis of aerosols by LIBS U. Panne and D. Hahn 6. Chemical imaging of surfaces using LIBS J. M. Vadillo and J. J. Laserna 7. Biomedical applications of LIBS H. H. Telle and O. Samek 8. LIBS for the analysis of pharmaceutical materials. S. Bechard and Y. Mouget 9. Cultural heritage applications of LIBS D. Anglos and J. C. Miller 10. Civilian and military environmental contamination studies using LIBS J. P. Singh, F. Y. Yueh, V. N. Rai, R. Harmon, S. Beaton, P. French, F. C. DeLucia, Jr., B. Peterson, K. L. McNesby and A. W. Miziolek 11. Industrial applications of LIBS R. Noll, V. Sturm, M. Stepputat, A. Whitehouse, J. Young and P. Evans 12. Resonance-enhanced LIBS N. H. Cheung 13. Short-pulse LIBS: fundamentals and applications R. E. Russo 14. High-speed, high resolution LIBS using diode-pumped solid state lasers H. Bette and R. Noll 15. LIBS using sequential laser pulses J. Pender, B. Pearman, J. Scaffidi, S. R. Goode and S. M. Angel 16. Micro LIBS technique P. Fichet, J-L, Lacour, D. Menut, P. Mauchien, A. Rivoallan, C. Fabre, J. Dubessy and M-C. Boiron 17. New spectral detectors for LIBS M. Sabsabi and V. Detalle 18. Spark-induced breakdown spectroscopy: a description of an electrically-generated LIBS-like process for elemental analysis of airborne particulates and solid samples A. J. R. Hunter and L. G. Piper.
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paper_title: Remote laser-induced plasma spectrometry for elemental analysis of samples of environmental interest

paper_content:

Remote laser-induced plasma spectrometry has been demonstrated as a valuable analytical tool both for qualitative inspection and quantitative determinations on environmental samples. For this objective, the pulsed radiation of a Q-switched Nd:YAG laser at 1064 nm has been used to produce a plasma in a remote sample, the light emission being collected under a coaxial open-path optical scheme and guided towards a spectrograph and then detected by an intensified CCD. A prospective study has been carried out to assess the suitability of the technique for the remote analysis of samples from a coastal scenario subjected to a high industrial activity. All the measurements have been done in the laboratory. Among the main factors influencing the analytical results, sample moisture and salinity, sample orientation and surface heterogeneity have been identified. The presence and distribution of Fe and Cr as a contaminant on sample surface has been quantified and discussed for samples including soil, rocks, and vegetation. At a stand-off distance of 12 m from the spectrometer to the sample, limits of detection in the order of 0.2% have been obtained for both elements.
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paper_title: Applications of laser-induced breakdown spectroscopy for geochemical and environmental analysis: A comprehensive review

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Abstract Applications of laser-induced breakdown spectroscopy (LIBS) have been growing rapidly and continue to be extended to a broad range of materials. This paper reviews recent application of LIBS for the analysis of geological and environmental materials, here termed "GEOLIBS" . Following a summary of fundamentals of the LIBS analytical technique and its potential for chemical analysis in real time, the history of the application of LIBS to the analysis of natural fluids, minerals, rocks, soils, sediments, and other natural materials is described.
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paper_title: Review: Applications of single-shot laser-induced breakdown spectroscopy

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As applications for laser-induced breakdown spectroscopy (LIBS) become more varied with a greater number of field and industrial LIBS systems developed and as the technique evolves to be more quantitative that qualitative, there is a more significant need for LIBS systems capable of analysis with the use of a single laser shot. In single-shot LIBS, a single laser pulse is used to form a single plasma for spectral analysis. In typical LIBS measurements, multiple laser pulses are formed and collected and an ensemble-averaged method is applied to the spectra. For some applications there is a need for rapid chemical analysis and/or non-destructive measurements; therefore, LIBS is performed using a single laser shot. This article reviews in brief several applications that demonstrate the applicability and need for single-shot LIBS.
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paper_title: Femtosecond Laser-Induced Breakdown Spectroscopy: Physics, Applications, and Perspectives

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Progress in technology and society continually places new demands on analytical science and more powerful and informative methods need to be developed. One among them is laser-induced breakdown spectroscopy (LIBS), sometimes also referred to as LIPS (laserinduced plasma spectroscopy). Typically, LIBS measurements are conducted with nanosecond time scale lasers. A review by Song et al.1 and two recently published books by Miziolek et al.2 and Cremers et al.3 give a good overview of instrumental developments in this area. However, new developments in laser technology have made ultra-short lasers available4 and have stimulated an interest in LIBS with ultra-short pulses. There are fundamental differences between the ablation processes of ultra-short ( 1 ps) and short ( 1 ps) pulses that result in different mechanisms of energy dissipation in the sample. In the case of ultra-short laser pulses, at the end of the laser pulse, only a very hot electron gas and a practically undisturbed lattice are found, which subsequently interact. However, for longer pulses above a certain energy threshold, the material undergoes transient changes in the thermodynamic states from solid, through liquid, into a plasma state.5,6 Based on this difference, consequences for the analytical performance of the method can be expected that in the future should lead to new aspects in instrumentation and applications of LIBS. The goal of this review is to summarize current knowledge of the instrumentation and physics of laser ablation with femtosecond lasers and to draw some conclusions concerning new possible applications that rely on these specific new features. It seems clear that even if a better performance in terms of analytical figures of merit compared to standard LIBS applications7 is found, a replacement of current technology cannot be expected soon due to the cost and complexity of chirped pulse amplification (CPA) laser systems. However, current trends in other fields of application of these laser systems, e.g., medical laser applications or material processing,8 may change this picture in the near future.
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paper_title: Laser-induced breakdown spectroscopy for quantitative analysis of copper in algae

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Laser-induced breakdown spectroscopy (LIBS) has been applied for quantitative analysis of Cu in algae plants, an issue of paramount importance for environmental monitoring. For the analysis with LIBS, algae were compacted into solid pellets with powdered calcium hydroxide addition as binder and a pulsed Nd:YAG laser was employed to produce the plasmas in air at atmospheric pressure. In this approach, atomic lines from traces of Cu were detected, as well as other major and minor elements. The plasma was characterized and a calibration curve was constructed with reference samples prepared with calcium hydroxide. The results obtained demonstrated the usefulness of the method for Cu monitoring in algae plants.
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paper_title: Laser Induced Breakdown Spectroscopy for Elemental Analysis in Environmental, Cultural Heritage and Space Applications: A Review of Methods and Results

paper_content:

Analytical applications of Laser Induced Breakdown Spectroscopy (LIBS), namely optical emission spectroscopy of laser-induced plasmas, have been constantly growing thanks to its intrinsic conceptual simplicity and versatility. Qualitative and quantitative analysis can be performed by LIBS both by drawing calibration lines and by using calibration-free methods and some of its features, so as fast multi-elemental response, micro-destructiveness, instrumentation portability, have rendered it particularly suitable for analytical applications in the field of environmental science, space exploration and cultural heritage. This review reports and discusses LIBS achievements in these areas and results obtained for soils and aqueous samples, meteorites and terrestrial samples simulating extraterrestrial planets, and cultural heritage samples, including buildings and objects of various kinds.
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paper_title: Laser-induced breakdown spectroscopy for analysis of plant materials: A review

paper_content:

Abstract Developments and contributions of laser-induced breakdown spectroscopy (LIBS) for the determination of elements in plant materials are reviewed. Several applications where the solid samples are interrogated by simply focusing the laser pulses directly onto a fresh or dried surface of leaves, roots, fruits, vegetables, wood and pollen are presented. For quantitative purposes aiming at plant nutrition diagnosis, the test sample presentation in the form of pressed pellets, prepared from clean, dried and properly ground/homogenized leaves, and the use of univariate or multivariate calibration strategies are revisited.
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paper_title: Comparing several atomic spectrometric methods to the super stars: special emphasis on laser induced breakdown spectrometry, LIBS, a future super star

paper_content:

The “super stars” of analytical atomic spectrometry are electrothermal atomization-atomic absorption spectrometry (ETA-AAS), inductively coupled plasma-atomic emission spectrometry (ICP-AES) and inductively coupled plasma-mass spectrometry (ICP-MS). Many other atomic spectrometric methods have been used to determine levels of elements present in solid, liquid and gaseous samples, but in most cases these other methods are inferior to the big three super star methods. The other atomic methods include glow discharge emission, absorption and mass spectrometric methods, laser excited fluorescence emission and ionization methods, and flame and microwave plasma emission and mass spectrometric methods. These “lesser” methods will be compared to the “super star” methods based on a number of figures of merit, including detection power, selectivity, multi-element capability, cost, applications, and “age” of the methods. The “age” of the method will be determined by a modification of the well-known Laitinen “Seven Ages of an Analytical Method” ::: (H.A. Laitinen, Anal. Chem., 1973, 45, 2305). Calculations will show that certain methods are capable of single atom detection, including several atomic absorption methods, as well as laser atomic ionization and fluorescence methods. The comparison of methods will indicate why the “super stars” of atomic spectrometric methods will continue to retain their status and what must be done for the lesser atomic methods to approach “super star” status. Certainly most of the lesser atomic spectrometric methods will have a limited place in the analytical arena. Because of the wide current interest and research activity, special emphasis will be placed on the technique of laser induced breakdown spectrometry (LIBS). Its current status and future developments will therefore be reviewed.
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paper_title: Application of laser-induced breakdown spectroscopy to the analysis of algal biomass for industrial biotechnology

paper_content:

Abstract We report on the application of laser-induced breakdown spectroscopy (LIBS) to the determination of elements distinctive in terms of their biological significance (such as potassium, magnesium, calcium, and sodium) 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 biological factors having an impact on them. Algae cells were suspended in liquid media or presented in a form of adherent cell mass on a surface (biofilm) and, consequently, characterized using their spectra. In our feasibility study we used three different experimental arrangements employing double-pulse LIBS technique in order to improve on analytical selectivity and sensitivity for potential industrial biotechnology applications, e.g. for monitoring of mass production of commercial biofuels, utilization in the food industry and control of the removal of heavy metal ions from industrial waste waters.
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paper_title: Quantitative analysis of toxic metals lead and cadmium in water jet by laser-induced breakdown spectroscopy

paper_content:

Laser-induced breakdown spectroscopy (LIBS) has been applied to the analysis of toxic metals Pb and Cd in Pb(NO3)2 and Cd(NO3)2.4H2O aqueous solutions, respectively. The plasma is generated by focusing a nanosecond Nd:YAG (λ=1064 nm) laser on the surface of liquid in the homemade liquid jet configuration. With an assumption of local thermodynamic equilibrium (LTE), calibration curves of Pb and Cd were obtained at different delay times between 1 to 5 μs. The temporal behavior of limit of detections (LOD) was investigated and it is shown that the minimum LODs for Pb and Cd are 4 and 68 parts in 106 (ppm), respectively. In order to demonstrate the correctness of the LTE assumption, plasma parameters including plasma temperature and electron density are evaluated, and it is shown that the LTE condition is satisfied at all delay times.
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paper_title: Determination of colloidal iron in water by laser-induced breakdown spectroscopy

paper_content:

Abstract Laser-induced breakdown spectroscopy was applied to the determination of FeO(OH) in water and successfully determined the concentration of the turbid solution down to a few ppm (μg ml −1 ). A Q-switched Nd:YAG laser, which delivered 8 ns pulses, was used as an excitation source. Cell-less measurement was achieved using a coaxial nozzle flow system which allowed to study the effect of ambient gas on emission intensity and decay lifetime of the breakdown plasma. Using helium gas and with a proper timing gate, the FeO(OH) concentration in water was determined in the ppm range.
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paper_title: Laser-Induced Breakdown Spectroscopy (LIBS), Part II: Review of Instrumental and Methodological Approaches to Material Analysis and Applications to Different Fields

paper_content:

The first part of this two-part review focused on the fundamental and diagnostics aspects of laser-induced plasmas, only touching briefly upon concepts such as sensitivity and detection limits and largely omitting any discussion of the vast panorama of the practical applications of the technique. Clearly a true LIBS community has emerged, which promises to quicken the pace of LIBS developments, applications, and implementations. With this second part, a more applied flavor is taken, and its intended goal is summarizing the current state-of-the-art of analytical LIBS, providing a contemporary snapshot of LIBS applications, and highlighting new directions in laser-induced breakdown spectroscopy, such as novel approaches, instrumental developments, and advanced use of chemometric tools. More specifically, we discuss instrumental and analytical approaches (e.g., double- and multi-pulse LIBS to improve the sensitivity), calibration-free approaches, hyphenated approaches in which techniques such as Raman and fluorescence are coupled with LIBS to increase sensitivity and information power, resonantly enhanced LIBS approaches, signal processing and optimization (e.g., signal-to-noise analysis), and finally applications. An attempt is made to provide an updated view of the role played by LIBS in the various fields, with emphasis on applications considered to be unique. We finally try to assess where LIBS is going as an analytical field, where in our opinion it should go, and what should still be done for consolidating the technique as a mature method of chemical analysis.
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paper_title: Application of laser-induced breakdown spectroscopy to in situ analysis of liquid samples

paper_content:

A realization of laser-induced breakdown spectroscopy for real-time, in situ and remote analysis of trace amounts in liquid samples is described, which is potentially applicable to the analysis of pollutants in water in harsh or difficult-to-reach environments. Most of the measurements were conducted using a fiber assembly that is capable of both delivering the laser light and collecting the light emitted from the micro plasma, up to about 30 m from the target area. Alternatively, a telescopic arrangement for line-of-sight measurements was employed, with a range of 3 to 5 m. For internal standardization and the generation of concentration calibration curves, reference lines of selected elements were used. In the majority of cases calibration against the matrix element hydrogen was employed using the H?, H?, and H? lines, but also spiking with selected reference species was utilized. In order to provide high reliability and repeatability in the analyses, we also measured plasma parameters such as electron density, plasma temperature, and line- shape functions, and determined their influence on the measurement results. Numerous elements, including a range of toxic heavy metals, have been measured over a wide range of concentrations (Al, Cr, Cu, Pb, Tc, U, and others). Limits of detection usually were in the range of a few parts per million; for several elements even lower concentrations could be measured.
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paper_title: Determination of an iron suspension in water by laser-induced breakdown spectroscopy with two sequential laser pulses.

paper_content:

We have applied laser-induced breakdown spectroscopy to quantitative analysis of colloidal and particulate iron in water. A coaxial sample flow apparatus developed in our previous work, which allowed us to control the atmosphere of laser-induced plasma, was used. Using sequential laser pulses from two Q-switched Nd:YAG lasers as excitation sources, the FeO(OH) concentration in the tens of ppb range was determined with an optimum interval between two laser pulses and an optimum delay time of a detector gate from the second pulse. The detection limit of Fe decreased substantially using two sequential laser pulse excitations: the 0.6 ppm limit of single pulse excitation to 16 ppb with sequential pulse excitation. The effects of the second laser pulse on the plasma emission were studied. The concentration of iron in fine particles in boiler water sampled from a commercially operated thermal power plant has been determined successfully by this method. The results show the capability of laser-induced breakdown spectroscopy in determining suspended colloidal and particulate impurities in a simple and quick way.
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paper_title: Investigation of laser-induced breakdown spectroscopy of a liquid jet

paper_content:

We investigate the feasibility of laser-induced breakdown spectroscopy for determination of heavy metal Pb in a Pb(NO3)2 aqueous solution by using a simple homemade vertical jet device and nanosecond laser pulses. Key experimental parameters that affect the analytical performance, such as delay of the time of observation, laser pulse energy, and liquid flow rate are optimized for the best limit of detection (LOD). The LOD was determined using Pb I emission at 405.781 nm, and after optimization, the 3σ LOD was found to be at the level of 60 ppm.
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paper_title: Study of laser-induced breakdown emission from liquid under double-pulse excitation.

paper_content:

The application of laser-induced breakdown spectroscopy to liquid samples, by use of a Nd:YAG laser in double-pulse excitation mode, is described. It is found that the line emission from a magnesium ion or atom is more than six times greater for double-pulse excitation than for single-pulse excitation. The effect of interpulse separation on the emission intensity of a magnesium ion and a neutral atom showed an optimum enhancement at a delay of 2.5–3 μs. The intensity of neutral atomic line emission dominates the ion emission from the plasma for higher interpulse (>10 μs) separation. A study of the temporal evolution of the line emission from the plasma shows that the background as well as line emission decays faster in double-pulse excitation than in single-pulse excitation. The enhancement in the emission seems to be dominated by an increase in the volume of the emitting gas. The limit of detection for a magnesium solution improved from 230 parts per billion (ppb) in single-pulse mode to 69 ppb in double-pulse mode.
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paper_title: Carbon emissions following 1.064 μm laser ablation of graphite and organic samples in ambient air

paper_content:

Laser ablation of graphite and organic samples is studied in the context of chemical analysis by laser-induced plasma spectroscopy. Ablation is performed using an Nd:YAG laser at 1.064 μm in ambient air at atmospheric pressure. Following ablation of graphite, we find results consistent with C2 (as well as C) being released directly from the target, and CN being formed later on by the interaction of C2 with atmospheric nitrogen (N2). In the case of organic compounds, we find a clear relationship between C2 emission from the plasma and the presence of aromatic rings (containing carbon–carbon double bonds) in the compounds.
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paper_title: Ultraviolet laser microplasma-gas chromatography detector: detection of species-specific fragment emission.

paper_content:

Characteristic laser-produced microplasma emissions from various simple carbon-containing vapors entrained in a He carrier gas have been observed and compared. A focused ArF (193-nm) excimer laser is used to induce microplasmas with modest pulse energies (15 mJ or less) in the effluent region of a gas chromatography capillary column. Strong atomic (C, H, O, Cl, and F) as well as molecular (C(2), CH, and CCI) emissions are observed with very high SNRs. A plasma emission survey indicates that different classes of molecule show unique spectra which make it relatively easy to distinguish one chemical class from another. These results suggest that a laser microplasma gas chromatography detector (LM-GCD) should offer additional discrimination/resolution for unknown sample gas mixture analysis. In addition, the LM-GCD exhibits a significant advantage over certain other GC detectors, like the widely used flame ionization detector, by readily detecting nonresponsive gases such as CO, CO(2), CCl(4) and Freons.
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paper_title: Application of laser-induced breakdown spectroscopy to the analysis of algal biomass for industrial biotechnology

paper_content:

Abstract We report on the application of laser-induced breakdown spectroscopy (LIBS) to the determination of elements distinctive in terms of their biological significance (such as potassium, magnesium, calcium, and sodium) 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 biological factors having an impact on them. Algae cells were suspended in liquid media or presented in a form of adherent cell mass on a surface (biofilm) and, consequently, characterized using their spectra. In our feasibility study we used three different experimental arrangements employing double-pulse LIBS technique in order to improve on analytical selectivity and sensitivity for potential industrial biotechnology applications, e.g. for monitoring of mass production of commercial biofuels, utilization in the food industry and control of the removal of heavy metal ions from industrial waste waters.
---
paper_title: Laser-Induced Breakdown Spectroscopy (LIBS), Part II: Review of Instrumental and Methodological Approaches to Material Analysis and Applications to Different Fields

paper_content:

The first part of this two-part review focused on the fundamental and diagnostics aspects of laser-induced plasmas, only touching briefly upon concepts such as sensitivity and detection limits and largely omitting any discussion of the vast panorama of the practical applications of the technique. Clearly a true LIBS community has emerged, which promises to quicken the pace of LIBS developments, applications, and implementations. With this second part, a more applied flavor is taken, and its intended goal is summarizing the current state-of-the-art of analytical LIBS, providing a contemporary snapshot of LIBS applications, and highlighting new directions in laser-induced breakdown spectroscopy, such as novel approaches, instrumental developments, and advanced use of chemometric tools. More specifically, we discuss instrumental and analytical approaches (e.g., double- and multi-pulse LIBS to improve the sensitivity), calibration-free approaches, hyphenated approaches in which techniques such as Raman and fluorescence are coupled with LIBS to increase sensitivity and information power, resonantly enhanced LIBS approaches, signal processing and optimization (e.g., signal-to-noise analysis), and finally applications. An attempt is made to provide an updated view of the role played by LIBS in the various fields, with emphasis on applications considered to be unique. We finally try to assess where LIBS is going as an analytical field, where in our opinion it should go, and what should still be done for consolidating the technique as a mature method of chemical analysis.
---
paper_title: Femtosecond time-resolved laser-induced breakdown spectroscopy for detection and identification of bacteria: A comparison to the nanosecond regime

paper_content:

Bacterial samples (Escherichia coli and Bacillus subtilis) have been analyzed by laser-induced breakdown spectroscopy (LIBS) using femtosecond pulses. We compare the obtained spectra with those resulting from the classical nanosecond LIBS. Specific features of femtosecond LIBS have been demonstrated, very attractive for analyzing biological sample: (i) a lower plasma temperature leading to negligible nitrogen and oxygen emissions from excited ambient air and a better contrast in detection of trace mineral species; and (ii) a specific ablation regime that favors intramolecular bonds emission with respect to atomic emission. A precise kinetic study of molecular band head intensities allows distinguishing the contribution of native CN bonds released by the sample from that due to carbon recombination with atmospheric nitrogen. Furthermore a sensitive detection of trace mineral elements provide specific spectral signature of different bacteria. An example is given for the Gram test provided by different magne...
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paper_title: Laser-induced breakdown spectroscopy (LIBS) : fundamenls and applications

paper_content:

Preface R. Russo and A. W. Miziolek 1. History and fundamentals of LIBS D. A. Cremers and L. J. Radziemski 2. Plasma morphology I. Schechter and V. Bulatov 3. From sample to signal in laser induced breakdown spectroscopy: a complex route to quantitative analysis E. Tognoni, V. Palleschi, M. Corsi, G. Cristoforetti, N. Omenetto, I. Gornushkin, B. W. Smith and J. D. Winefordner 4. Laser induced breakdown in gases: experiments and simulation C. G. Parigger 5. Analysis of aerosols by LIBS U. Panne and D. Hahn 6. Chemical imaging of surfaces using LIBS J. M. Vadillo and J. J. Laserna 7. Biomedical applications of LIBS H. H. Telle and O. Samek 8. LIBS for the analysis of pharmaceutical materials. S. Bechard and Y. Mouget 9. Cultural heritage applications of LIBS D. Anglos and J. C. Miller 10. Civilian and military environmental contamination studies using LIBS J. P. Singh, F. Y. Yueh, V. N. Rai, R. Harmon, S. Beaton, P. French, F. C. DeLucia, Jr., B. Peterson, K. L. McNesby and A. W. Miziolek 11. Industrial applications of LIBS R. Noll, V. Sturm, M. Stepputat, A. Whitehouse, J. Young and P. Evans 12. Resonance-enhanced LIBS N. H. Cheung 13. Short-pulse LIBS: fundamentals and applications R. E. Russo 14. High-speed, high resolution LIBS using diode-pumped solid state lasers H. Bette and R. Noll 15. LIBS using sequential laser pulses J. Pender, B. Pearman, J. Scaffidi, S. R. Goode and S. M. Angel 16. Micro LIBS technique P. Fichet, J-L, Lacour, D. Menut, P. Mauchien, A. Rivoallan, C. Fabre, J. Dubessy and M-C. Boiron 17. New spectral detectors for LIBS M. Sabsabi and V. Detalle 18. Spark-induced breakdown spectroscopy: a description of an electrically-generated LIBS-like process for elemental analysis of airborne particulates and solid samples A. J. R. Hunter and L. G. Piper.
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paper_title: Classification of vegetable oils based on their concentration of saturated fatty acids using laser induced breakdown spectroscopy (LIBS).

paper_content:

Spectrochemical analyses of organic liquid media such as vegetable oils and sweetened water were performed with the use of LIBS. The aim of this work is to study, on the basis of spectral analyses by LIBS technique of "Swan band" of C2 emitted by different vegetable oils in liquid phase, the characteristics of each organic media. Furthermore this paper proposes, as a classification, a single parameter that could be used to determine the concentration of saturated fatty acids of vegetable oils. A Nd:YAG operating at λ=532 nm and an energies per pulse of 30 mJ was focused onto the surface of the liquid in ambient air. Following ablation of vegetable oils and sweetened water, we find that vibrational bonds of C2 were released from the molecule containing carbon-carbon bonds linear. In the case of vegetable oils, we find a clear relationship between C2 emission from the plasma and the concentration of saturated fatty acids in the oil.
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paper_title: Laser-Induced Breakdown Spectroscopy of Liquids: Aqueous Solutions of Nickel and Chlorinated Hydrocarbons

paper_content:

Spectrochemical analyses of aqueous solutions containing nickel or the chlorinated hydrocarbons (CHCs) C2Cl4, CCl4, CHCl3, and C2HCl3 were performed with the use of laser-induced breakdown spectroscopy. A Nd:YAG laser operating at 60 mJ/pulse was focused onto the surface of the liquid. Elemental line intensities were monitored in the laser-produced plume as a function of analyte concentration to determine detection limits. The limits of detection for nickel in water were 36.4 +/- 5.4 mg/L and 18.0 +/- 3.8 mg/L for laser irradiation at 1.06 mu m and 355 nm, respectively. Ablation of pure CHCs at 355 nm produced extremely intense plasma emissions that primarily consisted of spectroscopic features attributed to CN, C3, H, N, and Cl. The spectra were structurally identical for all the CHCs except for differences in the intensities of various emission lines. With the use of emission from neutral atomic chlorine as an identifier for CHC contamination of water, no detectable traces of these elements were observed in saturated aqueous solutions. The detection limits for the CHCs were well above the saturation limits of CHC in water.
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paper_title: Quantitative molecular analysis with molecular bands emission using laser-induced breakdown spectroscopy and chemometrics

paper_content:

The present work describes the first quantitative molecular prediction using laser-induced molecular bands along with chemometrics. In addition, this spectroscopic procedure has demonstrated the first complete quantitative analysis utilizing traditionally insensitive elements for pharmaceutical formulations. Atomic LIBS requires certain sensitive elements, such as Cl, F, Br, S and P, in order to quantitate a specific organic compound in a complex matrix. Molecular LIBS has been demonstrated to be the first successful approach using atomic spectroscopy to evaluate a complex organic matrix. This procedure is also the first quantitative analysis using laser-induced molecular bands and chemometrics. We have successfully applied chemometrics to predict the formulation excipients and active pharmaceutical ingredient (API) in a complex pharmaceutical formulation. Using such an approach, we demonstrate that the accuracy for the API and a formulation lubricant, magnesium stearate, have less than 4% relative bias. The other formulation excipients such as Avicel® and lactose have been accurately predicted to have less than a 15% relative bias. Molecular LIBS and chemometrics have provided a novel approach for the quantitative analysis of several molecules that was not technically possible with the traditional atomic LIBS procedure, that required sensitive elements to be present in both API and formulation excipients.
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paper_title: Laser-induced breakdown spectroscopy of bacterial spores, molds, pollens, and protein: initial studies of discrimination potential

paper_content:

Laser-induced breakdown spectroscopy (LIBS) has been used to study bacterial spores, molds, pollens, and proteins. Biosamples were prepared and deposited onto porous silver substrates. LIBS data from the individual laser shots were analyzed by principal-components analysis and were found to contain adequate information to afford discrimination among the different biomaterials. Additional discrimination within the three bacilli studied appears feasible.
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paper_title: Detection of bacteria by time-resolved laser-induced breakdown spectroscopy.

paper_content:

A laser-induced breakdown spectroscopy technique for analyzing biological matter for the detection of biological hazards is investigated. Eight species were considered in our experiment: six bacteria and two pollens in pellet form. The experimental setup is described, then a cumulative intensity ratio is proposed as a quantitative criterion because of its linearity and reproducibility. Time-resolved laser-induced breakdown spectroscopy (TRELIBS) exhibits a good ability to differentiate among all these species, whatever the culture medium, the species or the strain. Thus we expect that TRELIBS will be a good candidate for a sensor of hazards either on surfaces or in ambient air.
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paper_title: Laser-Induced Breakdown Spectroscopy analysis of Bacteria: What Femtosecond Lasers Make Possible

paper_content:

Laser Induced Breakdown Spectroscopy spectra of bacteria, with nanosecond and femtosecond ablation, are compared. High sensitivity for mineral trace detections, larger intensity from molecular bands and precise kinetic study are among benefits using short pulses.
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paper_title: Elemental and radioactive analysis of commercially available seaweed.

paper_content:

Edible seaweed products have been used in many countries, specifically Japan, as a food item. Recently these products have become popular in the food industry because of a number of interesting medicinal properties that have been associated with certain edible marine algae. Very little control exists over the composition of these products, which could be contaminated with a number of agents including heavy metals and certain radioactive isotopes. Fifteen seaweed samples (six local samples from the coast of British Columbia, seven from Japan, one from Norway and one undisclosed) were obtained. All samples were analyzed for multiple elements, using ICP mass spectrometry and for radioactive constituents. It was found that six of eight imported seaweed products had concentrations of mercury orders of magnitude higher than the local products. Lead was found at somewhat higher concentrations in only one local product. Laminaria japonica had the highest level of iodine content followed by Laminaria setchellii from local sources. Only traces of cesium-137 were found in a product from Norway and radium-226 was found in a product from Japan. Arsenic levels were found to be elevated. In order to estimate the effect of these levels on health, one needs to address the bioavalability and the speciation of arsenic in these samples.
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paper_title: A general model for ontogenetic growth.

paper_content:

Several equations have been proposed to describe ontogenetic growth trajectories for organisms justified primarily on the goodness of fit rather than on any biological mechanism. Here, we derive a general quantitative model based on fundamental principles for the allocation of metabolic energy between maintenance of existing tissue and the production of new biomass. We thus predict the parameters governing growth curves from basic cellular properties and derive a single parameterless universal curve that describes the growth of many diverse species. The model provides the basis for deriving allometric relationships for growth rates and the timing of life history events.
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paper_title: High-resolution analysis of trace elements in crustose coralline algae from the North Atlantic and North Pacific by laser ablation ICP-MS

paper_content:

We have investigated the trace elemental composition in the skeleta of two specimens of attached-living ::: coralline algae of the species Clathromorphum compactum from the North Atlantic (Newfoundland) and ::: Clathromorphum nereostratum from the North Pacific/Bering Sea region (Amchitka Island, Aleutians). ::: Samples were analyzed using Laser Ablation-Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) ::: yielding for the first time continuous individual trace elemental records of up to 69 years in length. The ::: resulting algal Mg/Ca, Sr/Ca, U/Ca, and Ba/Ca ratios are reproducible within individual sample specimens. ::: Algal Mg/Ca ratios were additionally validated by electron microprobe analyses (Amchitka sample). Algal Sr/ ::: Ca, U/Ca, and Ba/Ca ratios were compared to algal Mg/Ca ratios, which previously have been shown to ::: reliably record sea surface temperature (SST). Ratios of Sr/Ca from both Clathromorphum species show a ::: strong positive correlation to temperature-dependent Mg/Ca ratios, implying that seawater temperature ::: plays an important role in the incorporation of Sr into algal calcite. Linear Sr/Ca-SST regressions have ::: provided positive, but weaker relationships as compared to Mg/Ca-SST relationships. Both, algal Mg/Ca and ::: Sr/Ca display clear seasonal cycles. Inverse correlations were found between algal Mg/Ca and U/Ca, Ba/Ca, ::: and correlations to SST are weaker than between Mg/Ca, Sr/Ca and SST. This suggests that the incorporation ::: of U and Ba is influenced by other factors aside from temperature
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paper_title: Freshening of the Alaska Coastal Current recorded by coralline algal Ba/Ca ratios

paper_content:

Arctic Ocean freshening can exert a controlling influence on global climate, triggering ::: strong feedbacks on ocean‐atmospheric processes and affecting the global cycling of the ::: world’s oceans. Glacier‐fed ocean currents such as the Alaska Coastal Current are ::: important sources of freshwater for the Bering Sea shelf, and may also influence the Arctic ::: Ocean freshwater budget. Instrumental data indicate a multiyear freshening episode of ::: the Alaska Coastal Current in the early 21st century. It is uncertain whether this freshening ::: is part of natural multidecadal climate variability or a unique feature of anthropogenically ::: induced warming. In order to answer this, a better understanding of past variations in ::: the Alaska Coastal Current is needed. However, continuous long‐term high‐resolution ::: observations of the Alaska Coastal Current have only been available for the last 2 decades. ::: In this study, specimens of the long‐lived crustose coralline alga Clathromorphum ::: nereostratum were collected within the pathway of the Alaska Coastal Current and utilized ::: as archives of past temperature and salinity. Results indicate that coralline algal Mg/Ca ::: ratios provide a 60 year record of sea surface temperatures and track changes of the Pacific ::: Decadal Oscillation, a pattern of decadal‐to‐multidecadal ocean‐atmosphere climate ::: variability centered over the North Pacific. Algal Ba/Ca ratios (used as indicators of coastal ::: freshwater runoff) are inversely correlated to instrumentally measured Alaska Coastal ::: Current salinity and record the period of freshening from 2001 to 2006. Similar multiyear ::: freshening events are not evident in the earlier portion of the 60 year Ba/Ca record. ::: This suggests that the 21st century freshening of the Alaska Coastal Current is a unique ::: feature related to increasing glacial melt and precipitation on mainland Alaska.
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paper_title: High-resolution Mg/Ca ratios in a coralline red alga as a proxy for Bering Sea temperature variations from 1902 to 1967

paper_content:

We present the first continuous, high-resolution record of Mg/Ca variations within an encrusting coralline red alga, Clathromorphum nereostratum, from Amchitka Island, Aleutian Islands. Mg/Ca ratios of individual growth increments were analyzed by measuring a singlepoint, electron-microprobe transect, yielding a resolution of ~15 samples/year and a 65-year record (1902–1967) of variations. Results show that Mg/Ca ratios in the high-Mg calcite algal framework display ::: pronounced annual cyclicity and archive late spring–late fall sea-surface temperatures (SST) corresponding to the main season of algal growth. Mg/Ca values correlate well to local SST, as well as to an air temperature record from the same region. High spatial correlation to large-scale SST variability in the subarctic North Pacific is observed, with patterns of strongest correlation following the direction of major oceanographic features that play a key role in the exchange of water masses between the North Pacific and the Bering Sea. Our data correlate well with a shorter Mg/Ca record from ability of the alga to reliably record regional environmental signals. In addition, Mg/Ca ratios relate well to a 29-year δ18O time series measured on the same sample, providing additional support for the use of Mg in coralline red algae as a paleotemperature proxy that, unlike algal-δ18O, is not influenced by salinity fluctuations. Moreover, electron microprobe–based analysis enables higher sampling resolution and faster analysis, thus providing a promising approach for future studies of longer C. nereostratum records and applications to other coralline species.
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paper_title: Development of a new sample pre-treatment procedure based on pressurized liquid extraction for the determination of metals in edible seaweed.

paper_content:

A new, simple, fast and automated method based on acetic acid-pressurized liquid extraction (PLE) has been developed for the simultaneous extraction of major and trace elements (As, Ca, Cd, Co, Cr, K, Mg, Mn, Na, Pb, Sr and Zn) from edible seaweeds. The target elements have been simultaneously determined by inductively coupled plasma-optical emission spectrometry (ICP-OES). The influence of several extraction parameters (e.g. acetic acid concentration, extraction temperature, extraction time, pressure, number of cycles, particle size and diatomaceous earth (DE) mass/sample mass ratio) on the efficiency of metal leaching has been evaluated. The results showed that metal extraction efficiency depends on the mass ratio of the dispersing agent mass and the sample. The optimized procedure consisted of the following conditions: acetic acid (0.75 M) as an extracting solution, 5 min of extraction time, one extraction cycle at room temperature at a pressure of 10.3 MPa and addition of a dispersing agent (at a ratio of 5:1 over the sample mass). The leaching procedure was completed after 7 min (5 min extraction time plus 1 min purge time plus 1 min end relief time). Limits of detection and quantification and repeatability of the over all procedure have been assessed. Method validation was performed analysing two seaweed reference materials (NIES-03 Chlorella Kessleri and NIES-09 Sargasso). The developed extraction method has been applied to red (Dulse and Nori), green (Sea Lettuce) and brown (Kombu, Wakame and Sea Spaghetti) edible seaweeds.
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paper_title: Coralline algal growth-increment widths archive North Atlantic climate variability

paper_content:

Over the past decade coralline algae have increasingly been used as archives of paleoclimate information. Encrusting coralline algae, which deposit annual growth increments in a high Mg-calcite skeleton, are amongst the longest-lived shallow marine organisms. In fact, a live-collected plant has recently been shown to have lived for at least 850 years based on radiometric dating. While a number of investigations have successfully used geochemical information of coralline algal skeletons to reconstruct sea surface temperatures, less attention has been paid to employ growth increment widths as a temperature proxy. Here we explore the relationship between growth and environmental parameters in Clathromorphum compactum collected in the subarctic Northwestern Atlantic. Results indicate that growth-increment widths of individual plants are poorly correlated with instrumental sea surface temperatures (SST). However, an averaged record of multiple growth increment-width time series from a regional network of C. compactum specimens up to 800 km apart reveals strong correlations with annual instrumental SST since 1950. Hence, similar to methods applied in dendrochronology, averaging of multiple sclerochronological records of coralline algae provides accurate climate information. A 115-year growth-increment width master chronology created from modern-collected and museum specimens is highly correlated to multidecadal variability seen in North Atlantic sea surface temperatures. Positive changes in algal growth anomalies record the well-documented regime shift and warming in the northwestern Atlantic during the 1990s. Large positive changes in algal growth anomalies were also present in the 1920s and 1930s, indicating that the impact of a concurrent large-scale regime shift throughout the North Atlantic was more strongly felt in the subarctic Northwestern Atlantic than previously thought, and may have even exceeded the 1990s event with respect to the magnitude of the warming.
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paper_title: Trace elements determination in edible seaweeds by an optimized and validated ICP-MS method

paper_content:

An optimized and validated inductively coupled plasma mass spectrometry (ICP-MS) method was used to analyze trace elements in seaweeds. Different volumes and rates of HNO3 and H2O2, digestion times, and microwave power levels were tested to ascertain the best conditions for sample digestion. Analytical mass and instrumental parameters were selected to assure accurate and precise determination of As, Cd, Co, Cr, Mo, Ni, Pb, Sb, Se, and V by ICP-MS. The method was optimized and validated using biological Certified Reference Materials. In addition, some samples of seaweeds (Porphyra and Laminaria) from France, Spain, Korea, and Japan were analyzed using the optimized method. Porphyra presented higher concentrations of most elements, except for As, than Laminaria. Seaweeds from Korea and Japan tended to display the highest concentrations of Pb and Cd. In contrast, Spanish and French samples showed the highest levels of some micro-elements essential to human nutrition.
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paper_title: Coralline algal Barium as indicator for 20th century northwestern North Atlantic surface ocean freshwater variability

paper_content:

During the past decades climate and freshwater dynamics in the northwestern North Atlantic have undergone major changes. Large-scale freshening episodes, related to polar freshwater pulses, have had a strong influence on ocean variability in this climatically important region. However, little is known about variability before 1950, mainly due to the lack of long-term high-resolution marine proxy archives. Here we present the first multidecadal-length records of annually resolved Ba/Ca variations from Northwest Atlantic coralline algae. We observe positive relationships between algal Ba/Ca ratios from two Newfoundland sites and salinity observations back to 1950. Both records capture episodical multi-year freshening events during the 20th century. Variability in algal Ba/Ca is sensitive to freshwater-induced changes in upper ocean stratification, which affect the transport of cold, Ba-enriched deep waters onto the shelf (highly stratified equals less Ba/Ca). Algal Ba/Ca ratios therefore may serve as a new resource for reconstructing past surface ocean freshwater changes.
---
paper_title: Mineral composition of edible seaweed Porphyra vietnamensis

paper_content:

Abstract Edible seaweed Porphyra vietnamensis growing along seven different localities of the Central West Coast of India was analyzed for mineral composition (Na, K, Ca, Mg, B, Pb, Cr, Co, Fe, Zn, Mn, Hg, Cu, As, Ni, Cd and Mo) by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The concentration ranges found for each sample, were as follows: Na, 24.5–65.6; K, 1.76–3.19, Ca, 1.40–6.12; Mg, 4.0–5.90 (mg/g d wt); Pb, 0.01–0.15; Cr, 0.13–0.22; Co, 0.06–0.20; Fe, 33.0–298; Zn, 0.93–3.27; Mn, 4.22–10.00; Hg, 0.01–0.04; Cu, 0.54–1.05; As, 1.24–1.83; Ni, 0.02–0.25; Cd, 0.14–0.55; Mo, 0.02–0.03 and B, 0.02–0.07 expressed in mg/100 g dry weight. Mineral composition of P. vietnamensis was found relatively higher as compared to the land vegetables as well as to other edible seaweeds, and it is in concurrence with the recent macrobiotic recommendation for western countries. It could therefore be used as food supplement as a spice to improve the nutritive value in the omnivorous diet.
---
paper_title: Light at work: the use of optical forces for particle manipulation, sorting, and analysis.

paper_content:

We review the combinations of optical micro-manipulation with other techniques and their classical and emerging applications to non-contact optical separation and sorting of micro- and nanoparticle suspensions, compositional and structural analysis of specimens, and quantification of force interactions at the microscopic scale. The review aims at inspiring researchers, especially those working outside the optical micro-manipulation field, to find new and interesting applications of these methods.
---
paper_title: Polarized Raman study of random copolymers of propylene with olefins

paper_content:

The polarized Raman spectroscopy is employed in the study of structural modifications in the films of isotactic polypropylene (PP) whose chain contains ethylene, 1-butene, 1-hexene, 1-octene, and 4-metyl-pentene-1, which represents an isomer of 1-hexene. It is demonstrated that the phase and conformational compositions of copolymer molecules depend on the comonomer content and the side-chain length of the second monomer. The content of the PP molecules in the helical conformation in the crystalline and amorphous phases of the copolymers monotonically decreases with increasing content of the second monomer. The decrease in the content of helical macromolecules in the crystalline phase is faster than the decrease in the amorphous phase. At a certain content of comonomers, the total content of the helical fragments decreases with increasing length of the side chain of the second monomer. The structures and Raman spectra of the copolymers of propylene with 1-hexene and 4-methyl-1-pentene are similar.
---
paper_title: The potential of Raman spectroscopy for the identification of biofilm formation by Staphylococcus epidermidis

paper_content:

We report on an investigation into a common problem in microbiology laboratories, which is associated with the difficulty of distinguishing/recognising different strains of the genus Staphylococcus. We demonstrate the potential of Raman spectroscopy as a rapid techniques allowing for the identification of different isolates for the detection of biofilm-positive and biofilm-negative Staphylococcus epidermidis strains. For this, the recorded spectra were interpreted using the approach of principal component analysis (PCA).
---
paper_title: In vivo Raman spectroscopy detects increased epidermal antioxidative potential with topically applied carotenoids

paper_content:

In the present study, the distribution of the carotenoids as a marker for the complete antioxidative potential in human skin was investigated before and after the topical application of carotenoids by in vivo Raman spectroscopy with an excitation wavelength of 785 nm. The carotenoid profile was assessed after a short term topical application in 4 healthy volunteers. In the untreated skin, the highest concentration of natural carotenoids was detected in different layers of the stratum corneum (SC) close to the skin surface. After topical application of carotenoids, an increase in the antioxidative potential in the skin could be observed. Topically applied carotenoids penetrate deep into the epidermis down to approximately 24 μm. This study supports the hypothesis that antioxidative substances are secreted via eccrine sweat glands and/or sebaceous glands to the skin surface. Subsequently they penetrate into the different layers of the SC.
---
paper_title: The holographic optical micro-manipulation system based on counter-propagating beams

paper_content:

We present a system employing a dynamic diffrac- tive optical element to control properties of two counter- propagating beams overlapping within a sample chamber. This system allows us to eliminate optical aberrations along both beam pathways and arbitrarily switch between various num- bers of laser beams and their spatial profiles (i.e. Gaussian, Laguerre-Gaussian, Bessel beams, etc.). We successfully tested various counter-propagating dual-beam configurations includ- ing optical manipulation of both high and low index particles in water or air, particle delivery in an optical conveyor belt and the formation of colloidal solitons by optical binding. Furthermore, we realized a novel optical mixer created by particles spiraling in counter-propagating interfering optical vortices and a new tool for optical tomography or localized spectroscopy enabling sterile contactless rotation and reorientation of a trapped living cell. CP Bessel beams in a form of optical conveyor belt
---
paper_title: Raman Microspectroscopy of Individual Algal Cells: Sensing Unsaturation of Storage Lipids in vivo

paper_content:

Algae are becoming a strategic source of fuels, food, feedstocks, and biologically active compounds. This potential has stimulated the development of innovative analytical methods focused on these microorganisms. Algal lipids are among the most promising potential products for fuels as well as for nutrition. The crucial parameter characterizing the algal lipids is the degree of unsaturation of the constituent fatty acids quantified by the iodine value. Here we demonstrate the capacity of the spatially resolved Raman microspectroscopy to determine the effective iodine value in lipid storage bodies of individual living algal cells. The Raman spectra were collected from three selected algal species immobilized in an agarose gel. Prior to immobilization, the algae were cultivated in the stationary phase inducing an overproduction of lipids. We employed the characteristic peaks in the Raman scattering spectra at 1,656 cm−1 (cis C═C stretching mode) and 1,445 cm−1 (CH2 scissoring mode) as the markers defining the ratio of unsaturated-to-saturated carbon-carbon bonds of the fatty acids in the algal lipids. These spectral features were first quantified for pure fatty acids of known iodine value. The resultant calibration curve was then used to calculate the effective iodine value of storage lipids in the living algal cells from their Raman spectra. We demonstrated that the iodine value differs significantly for the three studied algal species. Our spectroscopic estimations of the iodine value were validated using GC-MS measurements and an excellent agreement was found for the Trachydiscus minutus species. A good agreement was also found with the earlier published data on Botryococcus braunii. Thus, we propose that Raman microspectroscopy can become technique of choice in the rapidly expanding field of algal biotechnology.
---
paper_title: Investigation of denaturation of human serum albumin under action of cethyltrimethylammonium bromide by raman spectroscopy

paper_content:

The mechanism of denaturation of human serum albumin (HSA) under action of a cationic detergent—cethyltrimethylammonium bromide (CTAB) is investigated by Raman spectroscopy method. The percentage contents of α-helical segments in polypeptide chain of HSA at denaturation under action of different concentrations of CTAB at different values of pH is determined. It is shown, that more intensive denaturation of HSA under action of CTAB takes place at pH values, larger the isoelectric point of protein (pI 4.7).
---
paper_title: Monitoring of all hydrogen isotopologues at tritium laboratory Karlsruhe using Raman spectroscopy

paper_content:

We have recorded Raman spectra for all hydrogen isotopologues, using a CW Nd:YVO4 laser (5 W output power at 532 nm) and a high-throughput (f/1.8) spectrograph coupled to a Peltier-cooled (200 K) CCD-array detector (512 × 2048 pixels). A (static) gas cell was used in all measurements. We investigated (i) “pure” fillings of the homonuclear isotopologues H2, D2, and T2; (ii) equilibrated binary fillings of H2 + D2, H2 + T2, and D2 + T2, thus providing the heteronuclear isotopologues HD, HT, and DT in a controlled manner; and (iii) general mixtures containing all isotopologues at varying concentration levels. Cell fillings within the total pressure range 13–985 mbar were studied, in order to determine the dynamic range of the Raman system and the detection limits for all isotopologues. Spectra were recorded for an accumulation period of 1000 s. The preliminary data evaluation was based on simple peak-height analysis of the ro-vibrational Q1-branches, yielding 3σ measurement sensitivities of 5 × 10−3, 7 × 10−3, and 25 × 10−3 mbar for the tritium-containing isotopologues T2, DT, and HT, respectively. These three isotopologues are the relevant ones for the KATRIN experiment and in the ITER fusion fuel cycle. While the measurement reported here were carried out with static-gas fillings, the cells are also ready for use with flowing-gas samples.
---
paper_title: Characterization of oil-producing microalgae using Raman spectroscopy

paper_content:

Raman spectroscopy offers a powerful alternative analytical method for the detection and identification of lipids/oil in biological samples, such as algae and fish. Recent research in the authors' groups, and experimental data only very recently published by us and a few other groups suggest that Raman spectroscopy can be exploited in instances where fast and accurate determination of the iodine value (associated with the degree of lipid unsaturation) is required. Here the current status of Raman spectroscopy applications on algae is reviewed, and particular attention is given to the efforts of identifying and selecting oil-rich algal strains for the potential mass production of commercial biofuels and for utilization in the food industry.
---
paper_title: Optical trapping of microalgae at 735-1064 nm: photodamage assessment.

paper_content:

Living microalgal cells differ from other cells that are used as objects for optical micromanipulation, in that they have strong light absorption in the visible range, and by the fact that their reaction centers are susceptible to photodamage. We trapped cells of the microalga Trachydiscus minutus using optical tweezers with laser wavelengths in the range from 735 nm to 1064 nm. The exposure to high photon flux density caused photodamage that was strongly wavelength dependent. The photochemical activity before and after exposure was assessed using a pulse amplitude modulation (PAM) technique. The photochemical activity was significantly and irreversibly suppressed by a 30s exposure to incident radiation at 735, 785, and 835 nm at a power of 25 mW. Irradiance at 885, 935 and 1064 nm had negligible effect at the same power. At a wavelength 1064 nm, a trapping power up to 218 mW caused no observable photodamage.
---
paper_title: In vivo prediction of the nutrient status of individual microalgal cells using Raman microspectroscopy.

paper_content:

An in vivo method for predicting the nutrient status of individual algal cells using Raman microspectroscopy is described. Raman spectra of cells using 780 nm laser excitation show enhanced bands mainly attributable to chlorophyll a and beta-carotene. The relative intensities of chlorophyll a and beta-carotene bands changed under nitrogen limitation, with chlorophyll a bands becoming less intense and beta-carotene bands more prominent. Although spectra from N-replete and N-starved cell populations varied, each distribution was distinct enough such that multivariate classification methods, such as partial least squares discriminant analysis, could accurately predict the nutrient status of the cells from the Raman spectral data.
---
paper_title: Micro-Raman spectroscopy of algae: Composition analysis and fluorescence background behavior

paper_content:

Preliminary feasibility studies were performed using Stokes Raman scattering for compositional analysis of algae. Two algal species, Chlorella sorokiniana (UTEX #1230) and Neochloris oleoabundans (UTEX #1185), were chosen for this study. Both species were considered to be candidates for biofuel production. Raman signals due to storage lipids (specifically triglycerides) were clearly identified in the nitrogen-starved C. sorokiniana and N. oleoabundans, but not in their healthy counterparts. On the other hand, signals resulting from the carotenoids were found to be present in all of the samples. Composition mapping was conducted in which Raman spectra were acquired from a dense sequence of locations over a small region of interest. The spectra obtained for the mapping images were filtered for the wavelengths of characteristic peaks that correspond to components of interest (i.e., triglyceride or carotenoid). The locations of the components of interest could be identified by the high intensity areas in the composition maps. Finally, the time evolution of fluorescence background was observed while acquiring Raman signals from the algae. The time dependence of fluorescence background is characterized by a general power law decay interrupted by sudden high intensity fluorescence events. The decreasing trend is likely a result of photo-bleaching of cell pigments due to prolonged intense laser exposure, while the sudden high intensity fluorescence events are not understood.
---
paper_title: Raman Spectroscopy and Related Techniques in Biomedicine

paper_content:

In this review we describe label-free optical spectroscopy techniques which are able to non-invasively measure the (bio)chemistry in biological systems. Raman spectroscopy uses visible or near-infrared light to measure a spectrum of vibrational bonds in seconds. Coherent anti-Stokes Raman (CARS) microscopy and stimulated Raman loss (SRL) microscopy are orders of magnitude more efficient than Raman spectroscopy, and are able to acquire high quality chemically-specific images in seconds. We discuss the benefits and limitations of all techniques, with particular emphasis on applications in biomedicine—both in vivo (using fiber endoscopes) and in vitro (in optical microscopes).
---
paper_title: Review Raman based imaging in biological application- a perspective

paper_content:

Received 16 April 2012 Revised 08 June 2012 Accepted 19 July 2012 Early online 25 July 2012 Print 31 August 2012 Imaging by means of Raman spectroscopy has emerged as a powerful technique in the study of various chemical processes occurring in biology. This technique is non-invasive, label-free, capable of providing molecular identity and can be performed in robust conditions. However, one major drawback is its inherently weak signal. The ways to overcome this issue is to use Raman based methods e.g. Resonance Raman Spectroscopy (RRS), Surface Enhanced Raman Spectroscopy (SERS), Tip Enhanced Raman Spectroscopy (TERS). In this review, we gave a brief introduction of all these methods, with special emphasis on their recent advances and applications in various fields of life science. Corresponding author
---
paper_title: A Resonance Raman Method for the Rapid Detection and Identification of Algae in Water

paper_content:

Resonance Raman spectra are reported for aqueous suspensions of nine clones of marine plankton algae representing three classes, five genera, and seven species. Spectra are obtained easily either directly from culture or from samples concentrated by sedimentation. Spectra taken at 488 or 457.9 nm are of high quality and are sufficiently distinct to differentiate clones at the algal class level, and possibly also at the genus level. Strongest peaks occur near 1527 and 1158 cm−1 associated with ν(c = c) and ν(c – c) of carotenoid pigments, but information is contained in the entire region between 900 and 3000 cm−1 due to associated overtone and combination bands which can be assigned along with fundamental vibrations. Chlorophyll peaks also are quite pronounced. Spectra obtained using rapid flow techniques match those taken using slurries in sealed tubes if low laser power is used. The sensitivity and rapidity of the technique suggest that it may be useful in remote sensing applications.
---
paper_title: Raman microspectroscopy of algal lipid bodies: β-carotene quantification

paper_content:

Advanced optical instruments can serve for analysis and manipulation of individual living cells and their internal structures. We have used Raman microspectroscopic analysis for assessment of β-carotene concentration in algal lipid bodies (LBs) in vivo. Some algae contain β-carotene in high amounts in their LBs, including strains which are considered useful in biotechnology for lipid and pigment production. We have devised a simple method to measure the concentration of β-carotene in a mixture of algal storage lipids from the ratio of their Raman vibrations. This finding may allow fast acquisition of β-carotene concentration valuable, e.g., for Raman microspectroscopy assisted cell sorting for selection of the overproducing strains. Furthermore, we demonstrate that β-carotene concentration can be proportional to LB volume and light intensity during the cultivation. We combine optical manipulation and analysis on a microfluidic platform in order to achieve fast, effective, and non-invasive sorting based on the spectroscopic features of the individual living cells. The resultant apparatus could find its use in demanding biotechnological applications such as selection of rare natural mutants or artificially modified cells resulting from genetic manipulations.
---
paper_title: Raman Spectroscopy Cell-based Biosensors

paper_content:

One of the main challenges faced by biodetection systems is the ability to detect and identify a large range of toxins at low concentrations and in short times. Cell-based biosensors rely on detecting changes in cell behaviour, metabolism, or induction of cell death following exposure of live cells to toxic agents. Raman spectroscopy is a powerful technique for studying cellular biochemistry. Different toxic chemicals have different effects on living cells and induce different time-dependent biochemical changes related to cell death mechanisms. Cellular changes start with membrane receptor signalling leading to cytoplasmic shrinkage and nuclear fragmentation. The potential advantage of Raman spectroscopy cell-based systems is that they are not engineered to respond specifically to a single toxic agent but are free to react to many biologically active compounds. Raman spectroscopy biosensors can also provide additional information from the time-dependent changes of cellular biochemistry. Since no cell labelling or staining is required, the specific time dependent biochemical changes in the living cells can be used for the identification and quantification of the toxic agents. Thus, detection of biochemical changes of cells by Raman spectroscopy could overcome the limitations of other biosensor techniques, with respect to detection and discrimination of a large range of toxic agents. Further developments of this technique may also include integration of cellular microarrays for high throughput in vitro toxicological testing of pharmaceuticals and in situ monitoring of the growth of engineered tissues.
---
paper_title: Emerging concepts in deep Raman spectroscopy of biological tissue.

paper_content:

This article reviews emerging Raman techniques for deep, non-invasive characterisation of biological tissues. As generic analytical tools, the new methods pave the way for a host of new applications including non-invasive bone disease diagnosis, chemical characterisation of 'stone-like' materials in urology and cancer detection in a number of organs.
---
paper_title: Raman Spectroscopy of Algae: A Review

paper_content:

Algae are eukaryotic microorganisms which contain chlorophyll and are capable of photosynthesis. In various studies, Raman spectra have been used to identify a particular genus in a group of different types of algae. Each biomolecule has its own signature Raman spectrum. This characteristic signal can be used to identify and characterize the biomolecules in algae. Raman spectrum can be used to identify the components, determine the molecular structure and various properties of biomolecules in algae. With this view, this work presents a comprehensive review of current practices and advancements in Raman spectroscopy of Algae as well as in Raman spectroscopy of component biomolecules of different genus of Algae.
---
paper_title: Effects of pre-processing of Raman spectra onin vivo classification of nutrient status of microalgal cells

paper_content:

Raman spectra were obtained from cells of the chlorophyte unicellular eukaryotic alga Dunaliella tertiolecta, which had been grown either under nutrient-replete conditions or starved of nitrogen for 4 days. Spectra were rich in bands which could all be attributed to either chlorophyll a or β-carotene. A cursory examination of the differences between the spectra of replete and starved cells indicated a decline in chlorophyll a and an increase in β-carotene in chlorophytes. Unprocessed spectra showed pronounced baseline effects. A variety of pre-processing techniques were used in an attempt to visualise the spectral, and hence chemical, differences in the transformed data and perform classification based upon these differences. Six types of spectral pre-processing were compared: baseline correction with vector normalisation; Multiplicative Scatter Correction (MSC), Extended Multiplicative Signal Correction (EMSC); Standard Normal Variate (SNV); and vector normalised 1st and 2nd derivative spectra. Results for Soft Independent Modelling of Class Analogy (SIMCA) and Partial Least Squares (PLS) discriminant analysis were compared. All pre-processing methods allowed spectral differences between N-replete and N-starved spectra to be visualised, with derivatives and EMSC scoring the lowest RMSEC and RMSEV values with PLS and also the best overall classification results. SIMCA was not suited to classifying the nutrient classes under any of the pre-treatments, due to the small model distances involved.
---
paper_title: Raman Microspectroscopy of Individual Algal Cells: Sensing Unsaturation of Storage Lipids in vivo

paper_content:

Algae are becoming a strategic source of fuels, food, feedstocks, and biologically active compounds. This potential has stimulated the development of innovative analytical methods focused on these microorganisms. Algal lipids are among the most promising potential products for fuels as well as for nutrition. The crucial parameter characterizing the algal lipids is the degree of unsaturation of the constituent fatty acids quantified by the iodine value. Here we demonstrate the capacity of the spatially resolved Raman microspectroscopy to determine the effective iodine value in lipid storage bodies of individual living algal cells. The Raman spectra were collected from three selected algal species immobilized in an agarose gel. Prior to immobilization, the algae were cultivated in the stationary phase inducing an overproduction of lipids. We employed the characteristic peaks in the Raman scattering spectra at 1,656 cm−1 (cis C═C stretching mode) and 1,445 cm−1 (CH2 scissoring mode) as the markers defining the ratio of unsaturated-to-saturated carbon-carbon bonds of the fatty acids in the algal lipids. These spectral features were first quantified for pure fatty acids of known iodine value. The resultant calibration curve was then used to calculate the effective iodine value of storage lipids in the living algal cells from their Raman spectra. We demonstrated that the iodine value differs significantly for the three studied algal species. Our spectroscopic estimations of the iodine value were validated using GC-MS measurements and an excellent agreement was found for the Trachydiscus minutus species. A good agreement was also found with the earlier published data on Botryococcus braunii. Thus, we propose that Raman microspectroscopy can become technique of choice in the rapidly expanding field of algal biotechnology.
---
paper_title: Raman Spectroscopy Analysis of Botryococcene Hydrocarbons from the Green MicroalgaBotryococcus braunii

paper_content:

Abstract Botryococcus braunii, B race is a unique green microalga that produces large amounts of liquid hydrocarbons known as botryococcenes that can be used as a fuel for internal combustion engines. The simplest botryococcene (C30) is metabolized by methylation to give intermediates of C31, C32, C33, and C34, with C34 being the predominant botryococcene in some strains. In the present work we have used Raman spectroscopy to characterize the structure of botryococcenes in an attempt to identify and localize botryococcenes within B. braunii cells. The spectral region from 1600–1700 cm−1 showed ν(C=C) stretching bands specific for botryococcenes. Distinct botryococcene Raman bands at 1640 and 1647 cm−1 were assigned to the stretching of the C=C bond in the botryococcene branch and the exomethylene C=C bonds produced by the methylations, respectively. A Raman band at 1670 cm−1 was assigned to the backbone C=C bond stretching. Density function theory calculations were used to determine the Raman spectra of all botryococcenes to compare computed theoretical values with those observed. The analysis showed that the ν(C=C) stretching bands at 1647 and 1670 cm−1 are actually composed of several closely spaced bands arising from the six individual C=C bonds in the molecule. We also used confocal Raman microspectroscopy to map the presence and location of methylated botryococcenes within a colony of B. braunii cells based on the methylation-specific 1647 cm−1 botryococcene Raman shift.
---
paper_title: In vivo lipidomics using single-cell Raman spectroscopy

paper_content:

We describe a method for direct, quantitative, in vivo lipid profiling of oil-producing microalgae using single-cell laser-trapping Raman spectroscopy. This approach is demonstrated in the quantitative determination of the degree of unsaturation and transition temperatures of constituent lipids within microalgae. These properties are important markers for determining engine compatibility and performance metrics of algal biodiesel. We show that these factors can be directly measured from a single living microalgal cell held in place with an optical trap while simultaneously collecting Raman data. Cellular response to different growth conditions is monitored in real time. Our approach circumvents the need for lipid extraction and analysis that is both slow and invasive. Furthermore, this technique yields real-time chemical information in a label-free manner, thus eliminating the limitations of impermeability, toxicity, and specificity of the fluorescent probes common in currently used protocols. Although the single-cell Raman spectroscopy demonstrated here is focused on the study of the microalgal lipids with biofuel applications, the analytical capability and quantitation algorithms demonstrated are applicable to many different organisms and should prove useful for a diverse range of applications in lipidomics.
---
paper_title: In vivo prediction of the nutrient status of individual microalgal cells using Raman microspectroscopy.

paper_content:

An in vivo method for predicting the nutrient status of individual algal cells using Raman microspectroscopy is described. Raman spectra of cells using 780 nm laser excitation show enhanced bands mainly attributable to chlorophyll a and beta-carotene. The relative intensities of chlorophyll a and beta-carotene bands changed under nitrogen limitation, with chlorophyll a bands becoming less intense and beta-carotene bands more prominent. Although spectra from N-replete and N-starved cell populations varied, each distribution was distinct enough such that multivariate classification methods, such as partial least squares discriminant analysis, could accurately predict the nutrient status of the cells from the Raman spectral data.
---
paper_title: Micro-Raman spectroscopy of algae: Composition analysis and fluorescence background behavior

paper_content:

Preliminary feasibility studies were performed using Stokes Raman scattering for compositional analysis of algae. Two algal species, Chlorella sorokiniana (UTEX #1230) and Neochloris oleoabundans (UTEX #1185), were chosen for this study. Both species were considered to be candidates for biofuel production. Raman signals due to storage lipids (specifically triglycerides) were clearly identified in the nitrogen-starved C. sorokiniana and N. oleoabundans, but not in their healthy counterparts. On the other hand, signals resulting from the carotenoids were found to be present in all of the samples. Composition mapping was conducted in which Raman spectra were acquired from a dense sequence of locations over a small region of interest. The spectra obtained for the mapping images were filtered for the wavelengths of characteristic peaks that correspond to components of interest (i.e., triglyceride or carotenoid). The locations of the components of interest could be identified by the high intensity areas in the composition maps. Finally, the time evolution of fluorescence background was observed while acquiring Raman signals from the algae. The time dependence of fluorescence background is characterized by a general power law decay interrupted by sudden high intensity fluorescence events. The decreasing trend is likely a result of photo-bleaching of cell pigments due to prolonged intense laser exposure, while the sudden high intensity fluorescence events are not understood.
---
paper_title: Effects of pre-processing of Raman spectra onin vivo classification of nutrient status of microalgal cells

paper_content:

Raman spectra were obtained from cells of the chlorophyte unicellular eukaryotic alga Dunaliella tertiolecta, which had been grown either under nutrient-replete conditions or starved of nitrogen for 4 days. Spectra were rich in bands which could all be attributed to either chlorophyll a or β-carotene. A cursory examination of the differences between the spectra of replete and starved cells indicated a decline in chlorophyll a and an increase in β-carotene in chlorophytes. Unprocessed spectra showed pronounced baseline effects. A variety of pre-processing techniques were used in an attempt to visualise the spectral, and hence chemical, differences in the transformed data and perform classification based upon these differences. Six types of spectral pre-processing were compared: baseline correction with vector normalisation; Multiplicative Scatter Correction (MSC), Extended Multiplicative Signal Correction (EMSC); Standard Normal Variate (SNV); and vector normalised 1st and 2nd derivative spectra. Results for Soft Independent Modelling of Class Analogy (SIMCA) and Partial Least Squares (PLS) discriminant analysis were compared. All pre-processing methods allowed spectral differences between N-replete and N-starved spectra to be visualised, with derivatives and EMSC scoring the lowest RMSEC and RMSEV values with PLS and also the best overall classification results. SIMCA was not suited to classifying the nutrient classes under any of the pre-treatments, due to the small model distances involved.
---
paper_title: Raman Spectroscopy Analysis of Botryococcene Hydrocarbons from the Green MicroalgaBotryococcus braunii

paper_content:

Abstract Botryococcus braunii, B race is a unique green microalga that produces large amounts of liquid hydrocarbons known as botryococcenes that can be used as a fuel for internal combustion engines. The simplest botryococcene (C30) is metabolized by methylation to give intermediates of C31, C32, C33, and C34, with C34 being the predominant botryococcene in some strains. In the present work we have used Raman spectroscopy to characterize the structure of botryococcenes in an attempt to identify and localize botryococcenes within B. braunii cells. The spectral region from 1600–1700 cm−1 showed ν(C=C) stretching bands specific for botryococcenes. Distinct botryococcene Raman bands at 1640 and 1647 cm−1 were assigned to the stretching of the C=C bond in the botryococcene branch and the exomethylene C=C bonds produced by the methylations, respectively. A Raman band at 1670 cm−1 was assigned to the backbone C=C bond stretching. Density function theory calculations were used to determine the Raman spectra of all botryococcenes to compare computed theoretical values with those observed. The analysis showed that the ν(C=C) stretching bands at 1647 and 1670 cm−1 are actually composed of several closely spaced bands arising from the six individual C=C bonds in the molecule. We also used confocal Raman microspectroscopy to map the presence and location of methylated botryococcenes within a colony of B. braunii cells based on the methylation-specific 1647 cm−1 botryococcene Raman shift.
---
paper_title: In vivo lipidomics using single-cell Raman spectroscopy

paper_content:

We describe a method for direct, quantitative, in vivo lipid profiling of oil-producing microalgae using single-cell laser-trapping Raman spectroscopy. This approach is demonstrated in the quantitative determination of the degree of unsaturation and transition temperatures of constituent lipids within microalgae. These properties are important markers for determining engine compatibility and performance metrics of algal biodiesel. We show that these factors can be directly measured from a single living microalgal cell held in place with an optical trap while simultaneously collecting Raman data. Cellular response to different growth conditions is monitored in real time. Our approach circumvents the need for lipid extraction and analysis that is both slow and invasive. Furthermore, this technique yields real-time chemical information in a label-free manner, thus eliminating the limitations of impermeability, toxicity, and specificity of the fluorescent probes common in currently used protocols. Although the single-cell Raman spectroscopy demonstrated here is focused on the study of the microalgal lipids with biofuel applications, the analytical capability and quantitation algorithms demonstrated are applicable to many different organisms and should prove useful for a diverse range of applications in lipidomics.
---
paper_title: In vivo prediction of the nutrient status of individual microalgal cells using Raman microspectroscopy.

paper_content:

An in vivo method for predicting the nutrient status of individual algal cells using Raman microspectroscopy is described. Raman spectra of cells using 780 nm laser excitation show enhanced bands mainly attributable to chlorophyll a and beta-carotene. The relative intensities of chlorophyll a and beta-carotene bands changed under nitrogen limitation, with chlorophyll a bands becoming less intense and beta-carotene bands more prominent. Although spectra from N-replete and N-starved cell populations varied, each distribution was distinct enough such that multivariate classification methods, such as partial least squares discriminant analysis, could accurately predict the nutrient status of the cells from the Raman spectral data.
---
paper_title: Micro-Raman spectroscopy of algae: Composition analysis and fluorescence background behavior

paper_content:

Preliminary feasibility studies were performed using Stokes Raman scattering for compositional analysis of algae. Two algal species, Chlorella sorokiniana (UTEX #1230) and Neochloris oleoabundans (UTEX #1185), were chosen for this study. Both species were considered to be candidates for biofuel production. Raman signals due to storage lipids (specifically triglycerides) were clearly identified in the nitrogen-starved C. sorokiniana and N. oleoabundans, but not in their healthy counterparts. On the other hand, signals resulting from the carotenoids were found to be present in all of the samples. Composition mapping was conducted in which Raman spectra were acquired from a dense sequence of locations over a small region of interest. The spectra obtained for the mapping images were filtered for the wavelengths of characteristic peaks that correspond to components of interest (i.e., triglyceride or carotenoid). The locations of the components of interest could be identified by the high intensity areas in the composition maps. Finally, the time evolution of fluorescence background was observed while acquiring Raman signals from the algae. The time dependence of fluorescence background is characterized by a general power law decay interrupted by sudden high intensity fluorescence events. The decreasing trend is likely a result of photo-bleaching of cell pigments due to prolonged intense laser exposure, while the sudden high intensity fluorescence events are not understood.
---
paper_title: Effects of pre-processing of Raman spectra onin vivo classification of nutrient status of microalgal cells

paper_content:

Raman spectra were obtained from cells of the chlorophyte unicellular eukaryotic alga Dunaliella tertiolecta, which had been grown either under nutrient-replete conditions or starved of nitrogen for 4 days. Spectra were rich in bands which could all be attributed to either chlorophyll a or β-carotene. A cursory examination of the differences between the spectra of replete and starved cells indicated a decline in chlorophyll a and an increase in β-carotene in chlorophytes. Unprocessed spectra showed pronounced baseline effects. A variety of pre-processing techniques were used in an attempt to visualise the spectral, and hence chemical, differences in the transformed data and perform classification based upon these differences. Six types of spectral pre-processing were compared: baseline correction with vector normalisation; Multiplicative Scatter Correction (MSC), Extended Multiplicative Signal Correction (EMSC); Standard Normal Variate (SNV); and vector normalised 1st and 2nd derivative spectra. Results for Soft Independent Modelling of Class Analogy (SIMCA) and Partial Least Squares (PLS) discriminant analysis were compared. All pre-processing methods allowed spectral differences between N-replete and N-starved spectra to be visualised, with derivatives and EMSC scoring the lowest RMSEC and RMSEV values with PLS and also the best overall classification results. SIMCA was not suited to classifying the nutrient classes under any of the pre-treatments, due to the small model distances involved.
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paper_title: Raman Microspectroscopy of Individual Algal Cells: Sensing Unsaturation of Storage Lipids in vivo

paper_content:

Algae are becoming a strategic source of fuels, food, feedstocks, and biologically active compounds. This potential has stimulated the development of innovative analytical methods focused on these microorganisms. Algal lipids are among the most promising potential products for fuels as well as for nutrition. The crucial parameter characterizing the algal lipids is the degree of unsaturation of the constituent fatty acids quantified by the iodine value. Here we demonstrate the capacity of the spatially resolved Raman microspectroscopy to determine the effective iodine value in lipid storage bodies of individual living algal cells. The Raman spectra were collected from three selected algal species immobilized in an agarose gel. Prior to immobilization, the algae were cultivated in the stationary phase inducing an overproduction of lipids. We employed the characteristic peaks in the Raman scattering spectra at 1,656 cm−1 (cis C═C stretching mode) and 1,445 cm−1 (CH2 scissoring mode) as the markers defining the ratio of unsaturated-to-saturated carbon-carbon bonds of the fatty acids in the algal lipids. These spectral features were first quantified for pure fatty acids of known iodine value. The resultant calibration curve was then used to calculate the effective iodine value of storage lipids in the living algal cells from their Raman spectra. We demonstrated that the iodine value differs significantly for the three studied algal species. Our spectroscopic estimations of the iodine value were validated using GC-MS measurements and an excellent agreement was found for the Trachydiscus minutus species. A good agreement was also found with the earlier published data on Botryococcus braunii. Thus, we propose that Raman microspectroscopy can become technique of choice in the rapidly expanding field of algal biotechnology.
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paper_title: Raman Spectroscopy Analysis of Botryococcene Hydrocarbons from the Green MicroalgaBotryococcus braunii

paper_content:

Abstract Botryococcus braunii, B race is a unique green microalga that produces large amounts of liquid hydrocarbons known as botryococcenes that can be used as a fuel for internal combustion engines. The simplest botryococcene (C30) is metabolized by methylation to give intermediates of C31, C32, C33, and C34, with C34 being the predominant botryococcene in some strains. In the present work we have used Raman spectroscopy to characterize the structure of botryococcenes in an attempt to identify and localize botryococcenes within B. braunii cells. The spectral region from 1600–1700 cm−1 showed ν(C=C) stretching bands specific for botryococcenes. Distinct botryococcene Raman bands at 1640 and 1647 cm−1 were assigned to the stretching of the C=C bond in the botryococcene branch and the exomethylene C=C bonds produced by the methylations, respectively. A Raman band at 1670 cm−1 was assigned to the backbone C=C bond stretching. Density function theory calculations were used to determine the Raman spectra of all botryococcenes to compare computed theoretical values with those observed. The analysis showed that the ν(C=C) stretching bands at 1647 and 1670 cm−1 are actually composed of several closely spaced bands arising from the six individual C=C bonds in the molecule. We also used confocal Raman microspectroscopy to map the presence and location of methylated botryococcenes within a colony of B. braunii cells based on the methylation-specific 1647 cm−1 botryococcene Raman shift.
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paper_title: In vivo lipidomics using single-cell Raman spectroscopy

paper_content:

We describe a method for direct, quantitative, in vivo lipid profiling of oil-producing microalgae using single-cell laser-trapping Raman spectroscopy. This approach is demonstrated in the quantitative determination of the degree of unsaturation and transition temperatures of constituent lipids within microalgae. These properties are important markers for determining engine compatibility and performance metrics of algal biodiesel. We show that these factors can be directly measured from a single living microalgal cell held in place with an optical trap while simultaneously collecting Raman data. Cellular response to different growth conditions is monitored in real time. Our approach circumvents the need for lipid extraction and analysis that is both slow and invasive. Furthermore, this technique yields real-time chemical information in a label-free manner, thus eliminating the limitations of impermeability, toxicity, and specificity of the fluorescent probes common in currently used protocols. Although the single-cell Raman spectroscopy demonstrated here is focused on the study of the microalgal lipids with biofuel applications, the analytical capability and quantitation algorithms demonstrated are applicable to many different organisms and should prove useful for a diverse range of applications in lipidomics.
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paper_title: In vivo prediction of the nutrient status of individual microalgal cells using Raman microspectroscopy.

paper_content:

An in vivo method for predicting the nutrient status of individual algal cells using Raman microspectroscopy is described. Raman spectra of cells using 780 nm laser excitation show enhanced bands mainly attributable to chlorophyll a and beta-carotene. The relative intensities of chlorophyll a and beta-carotene bands changed under nitrogen limitation, with chlorophyll a bands becoming less intense and beta-carotene bands more prominent. Although spectra from N-replete and N-starved cell populations varied, each distribution was distinct enough such that multivariate classification methods, such as partial least squares discriminant analysis, could accurately predict the nutrient status of the cells from the Raman spectral data.
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paper_title: Chemometric prediction of alginate monomer composition: A comparative spectroscopic study using IR, Raman, NIR and NMR

paper_content:

Abstract The potential of using infrared (IR), Raman and near infrared (NIR) spectroscopy combined with chemometrics for reliable and rapid determination of the ratio of mannuronic and guluronic acid (M/G ratio) in commercial sodium alginate powders has been investigated. The reference method for quantification of the M/G ratio was solution-state 1 H nuclear magnetic resonance (NMR) spectroscopy. For a set of 100 commercial alginate powders with a M/G ratio range of 0.5–2.1 quantitative calibrations using partial least squares regression (PLSR) were developed and compared for the three spectroscopic methods. All three spectroscopic methods yielded models with prediction errors (RMSEP) of 0.08 and correlation coefficients between 0.96 and 0.97. However, the model based on extended inverted signal corrected (EISC) Raman spectra stood out by only using one PLS component for the prediction. The results are comparable to that of the experimental error of the reference method estimated to be between 0.01 and 0.08.
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paper_title: Application of laser-induced breakdown spectroscopy to the analysis of algal biomass for industrial biotechnology

paper_content:

Abstract We report on the application of laser-induced breakdown spectroscopy (LIBS) to the determination of elements distinctive in terms of their biological significance (such as potassium, magnesium, calcium, and sodium) 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 biological factors having an impact on them. Algae cells were suspended in liquid media or presented in a form of adherent cell mass on a surface (biofilm) and, consequently, characterized using their spectra. In our feasibility study we used three different experimental arrangements employing double-pulse LIBS technique in order to improve on analytical selectivity and sensitivity for potential industrial biotechnology applications, e.g. for monitoring of mass production of commercial biofuels, utilization in the food industry and control of the removal of heavy metal ions from industrial waste waters.
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paper_title: Comparison of green algae Cladophora sp. and Enteromorpha sp. as potential biomonitors of chemical elements in the southern Baltic.

paper_content:

The contents of Cd, Cu, Ni, Pb, Zn, Mn, K, Na, Ca and Mg were determined in the green algae Cladophora sp. from coastal and lagoonal waters of the southern Baltic. Factor analysis demonstrated spatial differences between concentration of chemical elements. The algae from the southern Baltic contained more Na and K while the anthropogenic impact of Cu, Pb and Zn was observed in the case of Cladophora sp. and Enteromorpha sp. from the Gulf of Gdansk at the vicinity of Gdynia. This area is exposed to emission of heavy metals from municipal and industrial sources with the main contribution of shipbuilding industry and seaport. The statistical evaluation of data has demonstrated that there exists a correlation between concentrations of Cu, Pb and Zn in both green algae collected at the same time and sampling sites of the Gulf of Gdansk. Our results show that in the case of absence of one species in the investigated area it is still possible to continue successfully the biomonitoring studies with its replacing by second one, i.e. Cladophora sp. by Enteromorpha sp. and vice versa; in consequence reliable results may be obtained.
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paper_title: INTERCOLONIAL VARIABILITY IN MACROMOLECULAR COMPOSITION IN P-STARVED AND P-REPLETE SCENEDESMUS POPULATIONS REVEALED BY INFRARED MICROSPECTROSCOPY(1).

paper_content:

Macromolecular variability in microalgal populations subject to different nutrient environments was investigated, using the chlorophyte alga Scenedesmus quadricauda (Turpin) Bréb. as a model organism. The large size of the four-cell coenobia in the strain used in this study (∼35 μm diameter) conveniently allowed high quality spectra to be obtained from individual coenobia using a laboratory-based Fourier transform infrared (FTIR) microscope with a conventional globar source of IR. By drawing sizable subpopulations of coenobia from two Scenedesmus cultures grown under either nutrient-replete or P-starved conditions, the population variability in macromolecular composition, and the effects of nutrient change upon this, could be estimated. On average, P-starved coenobia had higher carbohydrate and lower protein absorbance compared with P-replete coenobia. These parameters varied between coenobia with histograms of the ratio of absorbance of the largest protein and carbohydrate bands being Gaussian distributed. Distributions for the P-replete and P-starved subpopulations were nonoverlapping, with the difference in mean ratios for the two populations being statistically significant. Greater variance was observed in the P-starved subpopulation. In addition, multivariate models were developed using the spectral data, which could accurately predict the nutrient status of an independent individual coenobium, based on its FTIR spectrum. Partial least squares discriminant analysis (PLS-DA) was a better prediction method compared with soft independent modeling by class analogy (SIMCA).
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paper_title: Femtosecond time-resolved laser-induced breakdown spectroscopy for detection and identification of bacteria: A comparison to the nanosecond regime

paper_content:

Bacterial samples (Escherichia coli and Bacillus subtilis) have been analyzed by laser-induced breakdown spectroscopy (LIBS) using femtosecond pulses. We compare the obtained spectra with those resulting from the classical nanosecond LIBS. Specific features of femtosecond LIBS have been demonstrated, very attractive for analyzing biological sample: (i) a lower plasma temperature leading to negligible nitrogen and oxygen emissions from excited ambient air and a better contrast in detection of trace mineral species; and (ii) a specific ablation regime that favors intramolecular bonds emission with respect to atomic emission. A precise kinetic study of molecular band head intensities allows distinguishing the contribution of native CN bonds released by the sample from that due to carbon recombination with atmospheric nitrogen. Furthermore a sensitive detection of trace mineral elements provide specific spectral signature of different bacteria. An example is given for the Gram test provided by different magne...
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paper_title: Feasibility of Spectroscopic Characterization of Algal Lipids: Chemometric Correlation of NIR and FTIR Spectra with Exogenous Lipids in Algal Biomass

paper_content:

A large number of algal biofuels projects rely on a lipid screening technique for selecting a particular algal strain with which to work. We have developed a multivariate calibration model for predicting the levels of spiked neutral and polar lipids in microalgae, based on infrared (both near-infrared (NIR) and Fourier transform infrared (FTIR)) spectroscopy. The advantage of an infrared spectroscopic technique over traditional chemical methods is the direct, fast, and non-destructive nature of the screening method. This calibration model provides a fast and high-throughput method for determining lipid content, providing an alternative to laborious traditional wet chemical methods. We present data of a study based on nine levels of exogenous lipid spikes (between 1% and 3% (w/w)) of trilaurin as a triglyceride and phosphatidylcholine as a phospholipid model compound in lyophilized algal biomass. We used a chemometric approach to corrrelate the main spectral changes upon increasing phospholipid and triglyceride content in algal biomass collected from single species. A multivariate partial least squares (PLS) calibration model was built and improved upon with the addition of multiple species to the dataset. Our results show that NIR and FTIR spectra of biomass from four species can be used to accurately predict the levels of exogenously added lipids. It appears that the cross-species verification of the predictions is more accurate with the NIR models (R 2 = 0.969 and 0.951 and RMECV = 0.182 and 0.227% for trilaurin and phosphatidylcholine spike respectively), compared with FTIR (R 2 = 0.907 and 0.464 and RMECV = 0.302 and 0.767% for trilaurin and phosphatidylcholine spike, respectively). A fast high-throughput spectroscopic lipid fingerprinting method can be applied in a multitude of screening efforts that are ongoing in the microalgal research community.
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paper_title: The Use of Laser-Induced Breakdown Spectroscopy for Distinguishing between Bacterial Pathogen Species and Strains

paper_content:

Laser-induced breakdown spectroscopy (LIBS) was used in a blind study to successfully differentiate bacterial pathogens, both species and strain. The pathogens used for the study were chosen and prepared by one set of researchers. The LIBS data were collected and analyzed by another set of researchers. The latter researchers had no knowledge of the sample identities other than that (1) the first five of fifteen samples were unique (not replicates) and (2) the remaining ten samples consisted of two replicates of each of the first five samples. Using only chemometric analysis of the LIBS data, the ten replicate bacterial samples were successfully matched to each of the first five samples. The results of this blind study show it is possible to differentiate the bacterial pathogens Escherichia coli, three clonal methicillin-resistant Staphylococcus aureus (MRSA) strains, and one unrelated MRSA strain using LIBS. This is an important finding because it demonstrates that LIBS can be used to determine bacterial pathogen species within a defined sample set and can be used to differentiate between clonal relationships among strains of a single multiple-antibiotic-resistant bacterial species. Such a capability is important for the development of LIBS instruments for use in medical, water, and food safety applications.
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paper_title: A membrane basis for bacterial identification and discrimination using laser-induced breakdown spectroscopy

paper_content:

Nanosecond single-pulse laser-induced breakdown spectroscopy (LIBS) has been used to discriminate between two different genera of Gram-negative bacteria and between several strains of the Escherichia coli bacterium based on the relative concentration of trace inorganic elements in the bacteria. Of particular importance in all such studies to date has been the role of divalent cations, specifically Ca2+ and Mg2+, which are present in the membranes of Gram-negative bacteria and act to aggregate the highly polar lipopolysaccharide molecules. We have demonstrated that the source of emission from Ca and Mg atoms observed in LIBS plasmas from bacteria is at least partially located at the outer membrane by intentionally altering membrane biochemistry and correlating these changes with the observed changes in the LIBS spectra. The definitive assignment of some fraction of the LIBS emission to the outer membrane composition establishes a potential serological, or surface-antigen, basis for the laser-based identifi...
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paper_title: Spectral fingerprints of bacterial strains by laser-induced breakdown spectroscopy

paper_content:

Laser-induced breakdown spectroscopy (LIBS) is used to record the plasma emission for the colonies of vegetative cells or spores of five bacterial strains: Bacillus thuringiensis T34, Escherichia coli IHII/pHT315, Bacillus subtilis 168, Bacillus megaterium QM B1551, and Bacillus megaterium PV361. The major inorganic components of the bacterial samples, including Ca, Mn, K, Na, Fe, and phosphate, are clearly identified from the breakdown emission spectra. The bacterial spores accumulate a lot of calcium that shows strong LIBS emission at 393.7 and 396.9 nm. The diverse emissions from the phosphate component at 588.1 and 588.7 nm provide a fingerprint for bacterial strains. The relative change of inclusions in the bacteria is clearly distinguished by two-dimensional charts of the bacterial components. The results demonstrate the potential of the LIBS method for the rapid and low false-positive classification of bacteria with minimum sample preparation.
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paper_title: Application of laser-induced breakdown spectroscopy to the analysis of algal biomass for industrial biotechnology

paper_content:

Abstract We report on the application of laser-induced breakdown spectroscopy (LIBS) to the determination of elements distinctive in terms of their biological significance (such as potassium, magnesium, calcium, and sodium) 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 biological factors having an impact on them. Algae cells were suspended in liquid media or presented in a form of adherent cell mass on a surface (biofilm) and, consequently, characterized using their spectra. In our feasibility study we used three different experimental arrangements employing double-pulse LIBS technique in order to improve on analytical selectivity and sensitivity for potential industrial biotechnology applications, e.g. for monitoring of mass production of commercial biofuels, utilization in the food industry and control of the removal of heavy metal ions from industrial waste waters.
---
paper_title: The Use of Laser-Induced Breakdown Spectroscopy for Distinguishing between Bacterial Pathogen Species and Strains

paper_content:

Laser-induced breakdown spectroscopy (LIBS) was used in a blind study to successfully differentiate bacterial pathogens, both species and strain. The pathogens used for the study were chosen and prepared by one set of researchers. The LIBS data were collected and analyzed by another set of researchers. The latter researchers had no knowledge of the sample identities other than that (1) the first five of fifteen samples were unique (not replicates) and (2) the remaining ten samples consisted of two replicates of each of the first five samples. Using only chemometric analysis of the LIBS data, the ten replicate bacterial samples were successfully matched to each of the first five samples. The results of this blind study show it is possible to differentiate the bacterial pathogens Escherichia coli, three clonal methicillin-resistant Staphylococcus aureus (MRSA) strains, and one unrelated MRSA strain using LIBS. This is an important finding because it demonstrates that LIBS can be used to determine bacterial pathogen species within a defined sample set and can be used to differentiate between clonal relationships among strains of a single multiple-antibiotic-resistant bacterial species. Such a capability is important for the development of LIBS instruments for use in medical, water, and food safety applications.
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Title: Algal Biomass Analysis by Laser-Based Analytical Techniques—A Review

Section 1: Introduction
Description 1: Provide an overview of the importance of algal biomass as an alternative energy source and its potential in various applications, including biofuels, bioremediation, and industrial uses. Highlight the challenges and opportunities in algal biomass research.

Section 2: Laser-Induced Breakdown Spectroscopy
Description 2: Detail the principles, advantages, and applications of LIBS for elemental analysis of algal samples. Discuss the specific challenges and methodologies for improving LIBS sensitivity and accuracy.

Section 3: Laser-Induced Breakdown Spectroscopy of Liquid Samples
Description 3: Explain the techniques and approaches for conducting LIBS on liquid samples, particularly algal suspensions. Highlight the recent developments and improvements in this area.

Section 4: Laser-Induced Breakdown Spectroscopy for Molecular Analysis
Description 4: Discuss the capability of LIBS to detect molecular bands in plasma emissions, the applications of this technique, and the potential of combining LIBS with other spectroscopic methods for more comprehensive analyses.

Section 5: Laser Ablation Inductively Coupled Plasma Based Techniques
Description 5: Describe the use of LA-ICP-MS and LA-ICP-OES for algal biomass analysis, emphasizing the advantages, procedures, and specific applications in spatially-resolved elemental analysis.

Section 6: Raman Spectroscopy
Description 6: Provide an overview of Raman spectroscopy, its principles, applications, and the specific benefits of this technique for analyzing the biochemical composition and lipid content of algal cells. Mention the integration with optical tweezers for non-destructive in vivo analysis.

Section 7: Chemometrics for the Recognition of Algal Strains
Description 7: Introduce the role of chemometric algorithms in analyzing spectroscopic data from algal samples. Explain how these algorithms are used for qualitative and quantitative analysis and classification of algal strains.

Section 8: Discrimination of Four Algal Strains by LIBS
Description 8: Present a case study on using LIBS combined with chemometric algorithms to discriminate between different algal strains. Describe the methodology, results, and implications of this research.

Section 9: Conclusions and Future Prospects
Description 9: Summarize the key findings of the review, emphasizing the potential and limitations of laser-based techniques for algal biomass analysis. Discuss future research directions and technological advancements needed to overcome current challenges.