Cell‐level modeling of nutrient competition on the growth of microalgal biomass

Growth of microalgal biomass is driven by nutrient availability but also by species competition for these nutrients. In this study, a modeling procedure has been developed by means of which impacts of nutrient competition on microalgae cells can be investigated on a cell‐level. The goal of this modeling is to enhance the understanding of nutrient competition in a given biological environment and to enable predicting the biomass' species composition. These models are compared with experimental data and empirical assessments published earlier in this journal. Because these simulations have been built on very generic assumptions, transferring this chemometric methodology to other cell types is straightforward. Copyright © 2014 John Wiley & Sons, Ltd.     

Monitoring chemical impacts on cell cultures by means of image analyses

Many chemical processes are involved in the interactions of living cells with their environment; however, monitoring such processes often requires sophisticated analyzers. In this study, a sensing strategy based on imaging techniques has been developed to (i) enable cell discrimination based on their physical appearance such as size and shape and (ii) to build predictive models that relate the measured cell appearance to chemical parameters in their environment. Both goals aim at innovative and straightforward sensing strategies for analyzing cell–environment interactions. Image analyses offer several advantages such as the use of simpler, more robust sensors and the omission of extensive sample/sensor preparations. Imaging can analyze numerous cells and thus gains a culture representative insight rather than a potentially nonrepresentative single‐cell response. As a proof‐of‐principle application, different species of microalgae cells have been exposed to various nutrient conditions. Microalgae are known to sensitively adapt to changing nutrient conditions and could potentially become biological “probes” for chemical shifts in ecosystems. Because of considerable spreads of cell size and shapes within one class, size and shape distributions have been derived from visible images of cell cultures. It is shown that the novel image analyses are capable of discriminating different cell species based on their cell shapes and sizes. It is also demonstrated that in conjunction with the recently introduced, nonlinear multivariate “predictor surfaces”, the nutrient availability has a quantifiable impact on the cell size distributions. In this application, predictor surfaces are somewhat more precise than partial least squares. Copyright © 2012 John Wiley & Sons, Ltd.     

Selection of Microalgae and Cyanobacteria Strains for Bicarbonate-Based Integrated Carbon Capture and Algae Production System

Using microalgae to capture CO₂ from flue gas is an ideal way to reduce CO₂ emission, but this is challenged by the high cost of carbon capture and transportation. To address this problem, a bicarbonate-based integrated carbon capture and algae production system (BICCAPS) has been proposed, in which bicarbonate is used for algae culture, and the regenerated carbonate from this process can be used to capture more CO₂. High-concentration bicarbonate is obligate for the BICCAPS. Thus, different strains of microalgae and cyanobacteria were tested in this study for their capability to grow in high-concentration NaHCO₃. The highest NaHCO₃ concentrations they are tolerant to were determined as 0.30 M for Synechocystis sp. PCC6803, 0.60 M for Cyanothece sp., 0.10 M for Chlorella sorokiniana, 0.60 M for Dunaliella salina, and 0.30 M for Dunaliella viridis and Dunaliella primolecta. In further study, biomass production from culture of D. primolecta in an Erlenmeyer flask with either 0.30 M NaHCO₃ or 2 % CO₂ bubbling was compared, and no significant difference was detected. This indicates BICCAPS can reach the same biomass productivity as regular CO₂ bubbling culture, and it is promising for future application.     

Production,Purification, and Study of the Amino Acid Composition of Microalgae Proteins

Microalgae are known to be rich in protein. In this study, we aim to investigate methods of producing and purifying proteins of 98 microalgae including Chlorella vulgaris, Arthrospira platensis, Nostoc sp., Dunaliella salina, and Pleurochrysis carterae (Baltic Sea). Therefore, we studied their amino acid composition and developed a two-stage protein concentrate purification method from the microalgae biomass. After an additional stage of purification, the mass fraction of protein substances with a molecular weight greater than 50 kDa in the protein concentrate isolated from the biomass of the microalga Dunaliella salina increased by 2.58 times as compared with the mass fraction before filtration. In the protein concentrate isolated from the biomass of the microalga Pleurochrysis cartera, the relative content of the fraction with a molecular weight greater than 50.0 kDa reached 82.4%, which was 2.43 times higher than the relative content of the same fractions in the protein concentrate isolated from this culture before the two-stage purification. The possibilities of large-scale industrial production of microalgae biomass and an expanded range of uses determine the need to search for highly productive protein strains of microalgae and to optimize the conditions for isolating amino acids from them.     

Mixed IR/Vis Two‐Dimensional Spectroscopy: Chemical Exchange beyond the Vibrational Lifetime and Sub‐ensemble Selective Photochemistry

Two‐dimensional exchange spectroscopy (2D EXSY) is a powerful method to study the interconversion (chemical exchange) of molecular species in equilibrium. This method has recently been realized in femtosecond 2D‐IR spectroscopy, dramatically increasing the time resolution. However, current implementations allow the EXSY signal (and therefore the chemical process of interest) only to be tracked during the lifetime (T₁) of the observed spectroscopic transition. This is a severe limitation, as typical vibrational T₁ are only a few ps. An IR/Vis pulse sequence is presented that overcomes this limit and makes the EXSY signal independent of T₁. The same pulse sequence allows to collect time‐resolved IR spectra after electronic excitation of a particular chemical species in a mixture of species with strongly overlapping UV/Vis spectra. Different photoreaction pathways and dynamics of coexisting isomers or of species involved in different intermolecular interactions can thus be revealed, even if the species cannot be isolated because they are in rapid equilibrium.     

Diazanaphthalenes: A 13C NMR investigation on the site of protonation and pKa values

The pH dependence of the ¹³C chemical shifts (δ) of the diazanaphthalenes has been recorded. From this dependence the pK_a values have been determined using the Henderson-Hasselbach equation. The change in ¹³C chemical shifts under the influence of nitrogen protonation (Δδ) has been predicted using the Δδ values of quinoline and isoquinoline. The correlation between observed and expected Δδ values of the symmetric diazanaphthalenes is very good. Assuming these changes in chemical shifts to be of general validity, the site of protonation in the asymmetric diazanaphthalenes has been determined by comparison of the expected Δδ values for α- and β-nitrogen protonation with the observed ones. The site of protonation for 1,6- and 1,7-naphthyridine is the β-nitrogen atom, whereas for cinnoline both monoprotonated species are present in a significant amount.     

NMR-Spectroscopic Investigations of Potentially Planarized Nonafulvenes

¹H- and ¹³C-NMR spectra of nonafulvene 1e and nonafulvenes 2 and 3 have been assigned, high-resolution ¹H-NMR spectra of 2 (600 MHz, Fig. 3) and of 3 (400 MHz, Fig. 2) have been analyzed, and the data are compared with those of other nonafulvenes (Tables 1–6). Generally speaking, according to their spectroscopic behavior, four classes of nonafulvenes (A–D) may be distinguished (Fig. 1). The investigation shows that compounds 1e and 3 belong to class A, being characterized by ¹H-chemical shifts around 6 ppm, strongly alternating ³J(H,H) and ¹³C chemical shifts in the range of 123 to 130 ppm, thus existing in the olefinic form with a non-planar nine-membered ring. On the other hand, 2 is the first nonafulvene of class D, being characterized by ¹H chemical shifts in the aromatic range, large ³J(H,H) values of the same size, and ¹³C chemical shifts around 110 ppm. Since NMR parameters are virtually not influenced by temperature (?50° to 50°) or solvents, it is concluded that 2 exclusively exists in the dipolar structure 2 ^± with a planarized nine-membered ring. According to Fig. 4, these classes (and their spectroscopic data) are linked by 10,10-bis(dimethylamino)nonafulvene ( 1c ; and its temperature-dependent NMR parameters): for 1c , a temperature-dependent equilibrium 1c?1c ^± had earlier been established.     

NMR Spectroscopic Study of the Self‐Aggregation of 3‐Hexen‐1,5‐diyne Derivatives

The self‐assembly of polycatenar molecules derived from 1,6‐diphenyl‐3,4‐dipropyl‐3‐hexen‐1,5‐diyne has been studied in detail by solution NMR spectroscopy. The analysis of the concentration‐ and temperature‐dependent evolution of the chemical shifts and the diffusion coefficients in [D₁₂]cyclohexane agrees well with an isodesmic model of association in this solvent. The association constants for the stacking and entropy and enthalpy of the process have been obtained. The driving force for the aggregation process is provided by a negative enthalpy (ΔH), which is partially compensated by a negative entropy (ΔS). A structural study of the self‐assembly in solution has been carried out with the help of NOESY NMR spectroscopic experiments.     

Cultivation of Chlorella protothecoides with Urban Wastewater in Continuous Photobioreactor: Biomass Productivity and Nutrient Removal

The capability to grow microalgae in nonsterilized wastewater is essential for an application of this technology in an actual industrial process. Batch experiments were carried out with the species in nonsterilized urban wastewater from local treatment plants to measure both the algal growth and the nutrient consumption. Chlorella protothecoides showed a high specific growth rate (about 1 day^?1), and no effects of bacterial contamination were observed. Then, this microalgae was grown in a continuous photobioreactor with CO₂–air aeration in order to verify the feasibility of an integrated process of the removal of nutrient from real wastewaters. Different residence times were tested, and biomass productivity and nutrients removal were measured. A maximum of microalgae productivity was found at around 0.8 day of residence time in agreement with theoretical expectation in the case of light-limited cultures. In addition, N-NH₄ and P-PO₄ removal rates were determined in order to model the kinetic of nutrients uptake. Results from batch and continuous experiments were used to propose an integrated process scheme of wastewater treatment at industrial scale including a section with C. protothecoides.     

29Si chemical shifts and additivity in pertrimethylsilylated O-methyl,O-benzyl,O-benzoyl and O-acetyl methyl β-D-xylopyranosides

²⁹Si chemical shifts in pertrimethylsilylated O-methyl, O-benzoyl, O-benzoyl and O-acetyl methyl β-D -xylopyranoside derivatives are reported. The chemical shifts in monotrimethylsilyl derivatives with the same O-substituents are in the order: δ(Si-2) > δ(Si-4) > δ(Si-3). When the chemical shifts in bis (trimethylsilyl) and tris(trimethylsilyl) derivatives are assigned according to this rule, all the chemical shifts agree well with a direct additivity rule. The only significant deviation from additivity occurs for methyl 2,4-di-O-benzoyl-3-O-trimethylsilyl-β-D -xylopyranoside, which has a different average conformation from the other compounds studied.     

Nitrogen-15 NMR evidence for the structures of N-9-H-Xanthen-9-yl- and N,N′-Di-9 H-xanthen-9-yl-ureas

A new monoxanthen‒9‒yl derivative of urea has been synthesized and the structure of this product (N‒9 H‒xanthen‒9‒ylurea) and that of the previously known N,N′‒di‒9 H‒xanthen‒9‒ylurea have been proved by ¹⁵N NMR and other spectroscopic techniques. A series of ¹³C and ¹⁵N labeled urea derivatives has been prepared and the utility of their ¹³C and ¹⁵N chemical shifts and coupling constants in the structural analysis of urea derivatives has been investigated.     

Temperature dependence of the carbon-13 chemical shifts of paraffinic hydrocarbons

A study of several linear and branched alkanes indicates that the temperature dependence of ¹³C chemical shifts is a complex phenomenon in which several non-additive effects may be operative. The chemical shift temperature coefficients dδ/dT do, however, reveal some systematic trends which could be helpful in the assignments of ¹³C resonances. An empirical equation is proposed (akin to that of Grant and Paul for ¹³C chemical shifts) which accurately correlates all the data obtained near ambient temperatures.     

Thermal characterisation of microalgae under slow pyrolysis conditions

Aquatic microalgae have high potential for production of bio-chemicals, liquid transport fuels and charcoal. Their main advantage over existing energy crops is that they have faster growth rates and do not compete with food production. In this study six species of microalgae (Tetraselmis chui, Chlorella like, Chlorella vulgaris, Chaetocerous muelleri, Dunaliella tertiolecta and Synechococcus) were selected, presenting a broad cross-section of physical characteristics and known behaviour under cultivation. The objective of this work was to ascertain differences in thermal conversion behaviour between the microalgae species under slow pyrolysis conditions.The samples were first analysed with a Computer Aided Thermal Analysis (CATA) technique at a standard heating rate of 10 °C/min. For all species, the energy required to achieve thermal conversion was found to be approximately 1 MJ/kg. Gas chromatography was then applied to measure the evolution of biogas compounds with temperature. The heat of combustion of the biogas compounds was estimated to vary significantly between species, ranging from 1.2 to 4.8 MJ/kg.Pyrolysis oil product yields were also estimated at 500 °C. The oils produced at this temperature were collected and their molecular weight distribution assessed by Matrix Assisted Laser Desorption/Ionisation (MALDI). The species were found to produce up to 43% by volume of bio-oils. In all samples the char fraction remained above one third of total sample weight.     

NMR studies of interionic interactions in N-methylformamide

The NMR chemical shifts of alkali and thallium(I) salts with various monovalent anions have been measured in N-methylformamide solution. Lithium-7 chemical shifts are virtually concentration and counter-ion independent, presumably due to an absence of direct cation-anion interactions. The sodium-23, potassium-39 and cesium-133 chemical shifts of the salts studied depend on the anion and vary linearly with the concentration. The observed behavior can be accounted for by the formation of collisional ion pairs. On the other hand, the thallium-205 chemical shifts of thallium(I) nitrate and perchlorate were anion-dependent and varied non-linearly with the salt concentration. These results are indicative of contact ion pair formation; formation constants were calculated to be 2.6±0.4 M ^–1 for TlNO ₃ and 1.7±0.5 M ^–1 for TlClO ₄ . The cesium-133 NMR spectra of several mixed electrolyte systems also have been measured in N-methylformamide solution. The¹³³Cs chemical shifts also change linearly with the concentrations of the salts added to 0.10 M CsI/NMF solutions. The influence of the anions on the chemical shifts is the same as that observed for cesium salts alone.     

Prototropic processes in benzaurins. <Superscript>19</Superscript>F and <Superscript>1</Superscript>H NMR spectra of fluoro- and methylsubstituted 4-hydroxyphenyl-diphenylcarbinols,related fuchsones and benzaurins

Tautomerism of benzaurins and hydration are studied. ¹H and ¹⁹F chemical shifts have been determined for a number of substituted 4-hydroxyphenyl-diphenyl carbinols containing fluorine in a 3-, 3*- or 4*-position, and for similar compounds containing additional methyl groups in a position of 3, 3** or 4**. The same data have been obtained for the fuchsones prepared by dehydration of the above carbinols. On this basis chemical shifts of fluorine in different positions have been evaluated as a monitor of the transformation of 4-hydroxyphenyl group to the semiquinone moiety. The ¹⁹F NMR can be used to monitor the transformation of 4**-fluorobenzaurin and the related 3,3*-disubstituted and 3,3*,5,5*-tetramethylsubstituted compounds to the corresponding carbinols due to the addition of a water molecule and to study the tautomerism of the two latter benzaurins as well as that of 3,3*,4**trifluorobenzaurin. Furthermore, fluorine and methyl group chemical shifts are sensitive to syn-anti-isomerism in substituted fuchsones.     

15N NMR studies of amino acids and their reaction products with formaldehyde

Natural abundance ¹⁵N NMR spectroscopy has been used to investigate the effect of pH on the ¹⁵N chemical shifts of lysine and of ε-hydroxymethyllysine. A computer calcualtion which fits the chemical shifts of both α-and ε-nitrogen atoms versus pH has been used to predict the pK_a values. ¹⁵N chemical shifts and some ¹J(¹⁵NH) values of some other amino acids and of their reaction products with formaldehyde are also reported.     

Investigation of substituent effects on the 1H and 13C NMR spectra of (Z)-N-(arylmethylene)-arylamine N-oxides (α,N-diaryl nitrones)

Substituent effects on the ¹H and ¹³C chemical shifts of 18 differently substituted (Z)-α,N-diaryl nitrones [N-(p-X-benzylidene)phenylamine N-oxides (Series I) and N-(benzylidene)-p-Y-phenylamine N-oxides (Series II)] have been obtained. A correlation has been found between the chemical shifts of the azomethine proton (H-α) and the Hammett σ parameters and the Swain and Lupton F and R parameters. Correlations of the chemical shifts of C-1 and C-4′ in Series I, and of C-α and C-1′ in Series II, with the same parameters have been investigated. In addition, the chemical shifts of the aromatic protons and carbons of the p-disubstituted (m-disubstituted in one case) benzene rings correlated with the appropriate substituent increments (Z_i). These correlations confirm the dual behaviour of the nitrone group and the presence of through-resonance in these nitrones.     

Nuclear magnetic resonance studies of 5,6-dicarboxy-2-norbornene derivatives

A detailed study has been made on the ¹H nuclear magnetic resonance (NMR) spectra of 5,6-dicarboxy-2-norbornene derivatives, whose chemical shifts and geminal, vicinal and long-range spin-spin coupling constants were determined. Each signal of the spectra of these derivatives shows a slight low-field shifts compared with norbornene itself. In the case of five methyl esters studied in this work, the signal of the 7s-proton remains at approximately the same position in all the compounds, but that of the 7a-proton is more variable. For the chemical shifts of the 7a-proton of the five methyl esters, an additive rule seems to hold: that the magnitude of the low-field shift, from the 7a-signal of norbornene itself, is obtained by adding each of the effects of the carboxyl and the carbomethoxy groups substituted at the endo-5-(or 6-) or the exo-5-(or 6-) positions of the norbornene ring. In most of the derivatives considered in this work, the spin-spin coupling constants, except those between bridgehead and bridged methylene protons, are about 1 to 2 Hz smaller than those of many other norbornene derivatives already studied. This fact seems to suggest that norbornenes, which have two bulky adjacent substituents at 5- and 6-positions, may suffer distortions in the norbornene ring.     

The 13C NMR spectra of nine ortho-substituted phenols

The ¹³C chemical shifts of nine ortho-substituted phenols have been determined in cyclohexane and in dimethyl sulfoxide. All data were acquired in duplicate upon c.w. and FT instrumentation and both sets of data are presented. The chemical shifts at carbons 1, 2 and 3 are correlated with the parameter Q. Q has been defined as P/Ir³ where P is the polarizability of the adjacent C? X bond, I is the first ionization potential of the elements F, Cl, Br, I and H, and r is the C? X bond length. Experimental values of Q are available for other substituents. The field and resonance parameters of Swain and Lupton may be combined with Q to form a three part multiple regression correlation which is more exact than that with Q alone and which applies to all carbons in the aromatic ring. The results of this study suggest that only one value of the Q parameter is needed to characterize the behavior of the nitro group in these solvent systems. This conclusion is contrasted with earlier results of the effect of substituents on proton chemical shifts in these systems.     

Oil Accumulation via Heterotrophic/Mixotrophic <Emphasis Type="Italic">Chlorella protothecoides</Emphasis>

Microalgal oil is a potential energy source because it can be easily converted to fatty acid methyl ester or hydrocarbon type of diesel, and it is produced with relatively higher productivity compared with oil from plants and animals. Heterotrophic growth of microalgae is superior due to its high final product concentration; however, the cost of the raw materials is unacceptable if sugar is utilized as the carbon source. The aim of this study is to optimize the lipid accumulation of Chlorella protothecoides by using carbon sources other than glucose in heterotrophic and mixotrophic cultures. Different factors such as different carbon sources, carbon to nitrogen ratio, initial pH level, salinity, and rotational speed are studied in affecting the cell growth and the oil accumulation. Our experiments revealed that the heterotrophic and mixotrophic cultures of C. protothecoides grew better than autotrophic cultures. C. protothecoides can grow on glycerol or acetate, as well as on glucose. Several stress factors were confirmed or discovered to significantly increase the lipid content of microalgae cells. The replacement of glycerol and acetate as carbon sources for microalgae cultivations provides potential for waste utilization: glycerol from biodiesel industry and acetate from biohydrogen production.