Application of the local regression method interval partial least-squares to the elucidation of protein secondary structure

The infrared amide bands are sensitive to the conformation of the polypeptide backbone of proteins. Since the backbone of proteins folds in complex spatial arrangements, the amide bands of these proteins result from the superimposition of vibration modes corresponding to the different types of structural motifs (alpha helices, beta sheets, etc.). Initially, band deconvolution techniques were applied to determine the secondary structure of proteins, i.e., the abundance of each structural motif in the polypeptide chain was directly related to the area of the suitable deconvolved vibration modes under the amide I band (1700-1600 cm(-1)). Recently, several multivariate regression methods have been used to predict the secondary structure of proteins as an alternative to the previous methods. They are based on establishing a relationship between a matrix of infrared protein spectra and another that includes their secondary structure, expressed as the fractions of the different structural motifs, determined from X-ray analysis. In this study, we investigated the use of the local regression method interval partial least-squares (iPLS) to seek improvements to the full-spectrum PLS and other regression methods. The local character of iPLS avoids the use of spectral regions that can introduce noise or that can be irrelevant for prediction and focuses on finding specific spectral ranges related to each secondary structure motif in all the proteins. This study has been applied to a representative protein data set with infrared spectra covering a large wavenumber range, including amides I-III bands (1700-1200 cm(-1)). iPLS has revealed new structural mode assignments related to less explored amide bands and has offered a satisfactory predictive ability using a small amount of selected specific spectral information.     

Effect of refining on pulp surface charge accessible to polydadmac and FTIR characteristic bands of high yield kraft fibres

Studies were carried out to determine the surface charge and total charge of refined softwood high yield kraft pulps at different levels by titration methods. The results showed that the surface charge was highly dependent on the degree of refining. However, the total fibre charge was not affected by refining. The change of the fibre surface charge during refining monitored using ATR-FTIR characteristic bands was within 1700-1300 cm(-1). Reliable PLS1 calibration models could be established to correlate the FTIR spectral data and the surface charge of the refined pulps.     

Phase change in amylose-water mixtures as seen by Fourier transform infrared

The phase content of amylose-water mixtures (0.7/0.3 w/w) has been analyzed by transmission Fourier transform infrared (FTIR) spectroscopy in the 1175-950 cm(-1) region. Spectra are obtained under three different conditions: (a) as a function of temperature (T) from 25 to 97 degrees C; (b) at room temperature (RT), after slow cycles of T; and (c) at RT after quenching. T(max), the maximum temperature in the cycle, ranges from 50 to 120 degrees C. The quality of the seven-band spectra allows for an unambiguous determination of each band area. Unexpectedly, slow cooling after different T(max) brings about wide changes in the spectra while quenching does not. Two jumps in the absorbance are found: one at 70 degrees C and the other above 105 degrees C. Previous work on slow calorimetry of amylose-water mixtures suggests that these temperatures correspond to the beginning and the end of the same physical phenomena that takes place slowly between these two temperatures-namely the dissolution of the strained network phase. The spectra have two distinct regions, the low wavenumber region (1078-950 cm(-1)) and the high wavenumber region (1175-1078 cm(-1)). A distinct gain in the integrated absorbance of the 1175-1078 cm(-1) region at the expanse of that of the 1078-950 cm(-1) region when T(max) increases is interpreted as a change from strained to unstrained environments. A nonequilibrium state between the chains is a strained environment. In light of the (13)C NMR evaluation of the change of molecular order with T, the observed changes of the ir spectra could correspond to a transformation of a network of double helices into freer chains, possibly single helices. The present in-depth analysis of equilibrium or near equilibrium states by FTIR can serve to understand, through in situ spectra, molecular mechanisms during the gelation/crystallization of amylose and other gel-forming polymers.     

Electrochemically induced FTIR difference spectroscopy in the mid- to far infrared (200 microm) domain: a new setup for the analysis of metal-ligand interactions in redox proteins

We report the setup of an electrochemical cell with chemical-vapor deposition diamond windows and the use of a Bruker 66 SX FTIR spectrometer equipped with DTGS and Si-bolometer detectors and KBr and mylar beam splitters, to record on the same sample, FTIR difference spectra corresponding to the structural changes associated with the change in redox state of active sites in proteins in the whole 1800-50 cm(-1) region. With cytochrome c we show that reliable reduced-minus-oxidized FTIR difference spectra are obtained, which correspond to single molecular vibrations. Redox-sensitive IR modes of the cytochrome c are detected until 140 cm(-1) with a good signal to noise. This new setup is promising to analyze the infrared spectral region where metal-ligand vibrations are expected to contribute and to extend the analysis of vibrational properties to metal sites or redox states not accessible to (resonance) Raman spectroscopy.     

Multivariate Analysis of Phenol in Freeze-Dried and Spray-Dried Insulin Formulations by NIR and FTIR

Dehydration is a commonly used method to stabilise protein formulations. Upon dehydration, there is a significant risk the composition of the formulation will change especially if the protein formulation contains volatile compounds. Phenol is often used as excipient in insulin formulations, stabilising the insulin hexamer by changing the secondary structure. We have previously shown that it is possible to maintain this structural change after drying. The aim of this study was to evaluate the residual phenol content in spray-dried and freeze-dried insulin formulations by Fourier transform infrared (FTIR) spectroscopy and near infrared (NIR) spectroscopy using multivariate data analysis. A principal component analysis (PCA) and partial least squares (PLS) projections were used to analyse spectral data. After drying, there was a difference between the two drying methods in the phenol/insulin ratio and the water content of the dried samples. The spray-dried samples contained more water and less phenol compared with the freeze-dried samples. For the FTIR spectra, the best model used one PLS component to describe the phenol/insulin ratio in the powders, and was based on the second derivative pre-treated spectra in the 850–650 cm⁻¹ region. The best PLS model based on the NIR spectra utilised three PLS components to describe the phenol/insulin ratio and was based on the standard normal variate transformed spectra in the 6,200–5,800 cm⁻¹ region. The root mean square error of cross validation was 0.69% and 0.60% (w/w) for the models based on the FTIR and NIR spectra, respectively. In general, both methods were suitable for phenol quantification in dried phenol/insulin samples.     

Low temperature FTIR spectroscopy provides new insights in the pH-dependent proton pathway of proteorhodopsin

In the presented study the low pH photocycle of proteorhodopsin is extensively investigated by means of low temperature FTIR spectroscopy. Besides the already well-known characteristics of the all-trans and 13-cis retinal vibrations the 77K difference spectrum at pH 5.1 shows an additional negative signal at 1744 cm(-1) which is interpreted as indicator for the L state. The subsequent photocycle steps are investigated at temperatures higher than 200K. The combination of visible and FTIR spectroscopy enabled us to observe that the deprotonation of the Schiff base is linked to the protonation of an Asp or Glu side chain - the new proton acceptor under acidic conditions. The difference spectra of the late intermediates are characterized by large amide I changes and two further bands ((-)1751 cm(-1)/(+)1725 cm(-1)) in the spectral region of the Asp/Glu ν(C=O) vibrations. The band position of the negative signature points to a transient deprotonation of Asp-97. In addition, the pH dependence of the acidic photocycle was investigated. The difference spectra at pH 5.5 show distinct differences connected to changes in the protonation state of key residues. Based on our data we propose a three-state model that explains the complex pH dependence of PR.     

Biospectroscopy to metabolically profile biomolecular structure: a multistage approach linking computational analysis with biomarkers

Biospectroscopy is employed to derive absorbance spectra representative of biomolecules present in biological samples. The mid-infrared region (λ = 2.5 μm-25 μm) is absorbed to give a biochemical-cell fingerprint (v = 1800-900 cm(-1)). Cellular material produces complex spectra due to the variety of chemical bonds present. The complexity and size of spectral data sets warrant multivariate analysis for data reduction, interpretation, and classification. Various multivariate analyses are available including principal component analysis (PCA), partial least-squares (PLS), linear discriminant analysis (LDA), and evolving fuzzy rule-based classifier (eClass). Interpretation of both visual and numerical results facilitates biomarker identification, cell-type discrimination, and predictive and mechanistic understanding of cellular behavior. Biospectroscopy is a high-throughput nondestructive technology. A comparison of biomarkers/mechanistic knowledge determined from conventional approaches to biospectroscopy coupled with multivariate analysis often provides complementary answers and a novel approach for diagnosis of disease and cell biology.     

Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture

Transient expression of recombinant proteins in mammalian cell culture in a 100-L scale requires a large quantity of plasmid that is very labour intensive to achieve with shake flask cultures and commercially available plasmid purification kits. In this paper we describe a process for plasmid production in 100-mg scale. The fermentation is carried out in a 4-L fed-batch culture with a minimal medium. The detection of the end of batch and triggering the exponential (0.1 h(-1)) feed profile was unattended and controlled by Multi-fermenter Control System. A restricted specific growth rate in fed-batch culture increased the specific plasmid yield compared to batch cultures with minimal and rich media. This together with high biomass concentration (68-107 g L(-1) wet weight) achieves high volumetric yields of plasmid (95-277 mg L(-1) depending on the construct). The purification process consisted of alkaline lysis, lysate clarification and ultrafiltration, two-phase extraction with Triton X-114 for endotoxin removal, anion-exchange chromatography as a polishing step, ultrafiltration and sterile filtration. Both fermentation and purification processes were used without optimisation for production of four plasmids yielding from 39 to 163 mg of plasmids with endotoxin content of 2.5 EU mg(-1) or less.     

Vibrational modes of tyrosines in cytochrome c oxidase from Paracoccus denitrificans: FTIR and electrochemical studies on Tyr-D4-labeled and on Tyr280His and Tyr35Phe mutant enzymes

A combined electrochemical and FTIR spectroscopic approach was used to identify the vibrational modes of tyrosines in cytochrome c oxidase from Paracoccus denitrificans which change upon electron transfer and coupled proton transfer. Electrochemically induced FTIR difference spectra of the Tyr-D4-labeled cytochrome c oxidase reveal that only small contributions arise from the tyrosines. Contributions between 1600 and 1560 cm(-1) are attributed to nu8a/8b(CC) ring modes. The nu19(CC) ring mode for the protonated form of tyrosines is proposed to absorb with an uncommonly small signal at 1525-1518 cm(-1) and for the deprotonated form at 1496-1486 cm(-1), accompanied by the increase of the nu19(CC) ring mode of the Tyr-D(4)-labeled oxidase at approximately 1434 cm(-1). A signal at 1270 cm(-1) can be tentatively attributed to the nu7'a(CO) and delta(COH) mode of a protonated tyrosine. Uncommon absorptions, like the mode at 1524 cm(-1), indicate the involvement of Tyr280 in the spectra. Tyr280 is a crucial residue close to the binuclear center and is covalently bonded to His276. The possible changes of the spectral properties are discussed together with the absorbance spectra of tyrosine bound to histidine. The vibrational modes of Tyr280 are further analyzed in combination with the mutation to histidine, which is assumed to abolish the covalent bonding. The electrochemically induced FTIR difference spectra of the Tyr280His mutant point to a change in protonation state in the environment of the binuclear center. Together with an observed decrease of a signal at 1736 cm(-1), previously assigned to Glu278, a possible functional coupling is reflected. In direct comparison to the FTIR difference spectra of the D4-labeled compound and comparing the spectra at pH 7 and 4.8, the protonation state of Tyr280 is discussed. Furthermore, a detailed analysis of the mutant is presented, the FTIR spectra of the CO adduct revealing a partial loss of Cu(B). Electrochemical redox titrations reflect a downshift of the heme a3 midpoint potential by 95 +/- 10 mV. Another tyrosine identified to show redox dependent changes upon electron transfer is Tyr35, a residue in the proposed D-pathway of the cytochrome c oxidase.     

Monitoring cell cycle distributions in MCF-7 cells using near-field photothermal microspectroscopy

Microspectroscopic techniques such as Fourier transform infrared (FTIR) have played an important role in "fingerprinting" the biochemical composition of cellular components. Based on structure and function, complex biomolecules absorb energy in the mid-infrared (lambda = 2-20 microm) yielding characteristic vibrational infrared (IR) spectra. However, optical detection FTIR microspectroscopy may not be suitable for IR-absorbing sample materials. Photothermal microspectroscopy (PTMS) permits the direct measurement of heat generated as a result of sample material absorbing radiation. This approach generates true absorption spectra and is implemented by interfacing a scanning probe microscope and an FTIR spectrometer. Detection is performed using a near-field ultra-miniaturized temperature sensor. Employing PTMS, IR spectra of MCF-7 cells were examined in spectral regions (900-2000 cm(-1)) corresponding to proteins, DNA, RNA, glycoproteins, carbohydrates, lipids, and levels of protein phosphorylation. As a cell passes through the cell cycle, its nuclear material decondenses and condenses and this has led to ambiguity as to whether the intensity of such spectral regions may be associated with the G(1)-, S- or G(2)-phases of the cell cycle. Cultured cells were tracked over a time course known to correspond to marked alterations in cell-cycle distributions, as determined using flow cytometry. Experiments were carried out in the absence or presence of lindane, a pesticide known to induce G(1)-arrest in MCF-7 cells. Significant (P < 0.05) elevations in spectral intensities were associated with exponentially growing cell populations, predominantly in S-phase or G(2)-phase, compared to more quiescent populations predominantly in G(1)-phase. Increases in the absorption band at 970 cm(-1), associated with elevated protein phosphorylation, were observed in vibrational spectra of exponentially growing cell populations compared to those exhibiting a slowing in their growth kinetics. These results seem to suggest that intracellular bulk changes, associated with transit through the cell cycle, can be tracked using PTMS.     

Prediction of Cell Wall Properties and Response to Deconstruction Using Alkaline Pretreatment in Diverse Maize Genotypes Using Py-MBMS and NIR

In this work, we explore the ability of several characterization approaches for phenotyping to extract information about plant cell wall properties in diverse maize genotypes with the goal of identifying approaches that could be used to predict the plant’s response to deconstruction in a biomass-to-biofuel process. Specifically, a maize diversity panel was subjected to two high-throughput biomass characterization approaches, pyrolysis molecular beam mass spectrometry (py-MBMS) and near-infrared (NIR) spectroscopy, and chemometric models to predict a number of plant cell wall properties as well as enzymatic hydrolysis yields of glucose following either no pretreatment or with mild alkaline pretreatment. These were compared to multiple linear regression (MLR) models developed from quantified properties. We were able to demonstrate that direct correlations to specific mass spectrometry ions from pyrolysis as well as characteristic regions of the second derivative of the NIR spectrum regions were comparable in their predictive capability to partial least squares (PLS) models for p-coumarate content, while the direct correlation to the spectral data was superior to the PLS for Klason lignin content and guaiacyl monomer release by thioacidolysis as assessed by cross-validation. The PLS models for prediction of hydrolysis yields using either py-MBMS or NIR spectra were superior to MLR models based on quantified properties for unpretreated biomass. However, the PLS models using the two high-throughput characterization approaches could not predict hydrolysis following alkaline pretreatment while MLR models based on quantified properties could. This is likely a consequence of quantified properties including some assessments of pretreated biomass, while the py-MBMS and NIR only utilized untreated biomass.     

Infrared microscopy for the study of biological cell monolayers. I. Spectral effects of acetone and formalin fixation

Infrared spectroscopy of biological cell monolayers grown on surfaces is a poorly developed field. This is unfortunate because these monolayers have potential as biological sensors. Here we have used infrared microscopy, in both transmission and transflection geometries, to study air-dried Vero cell monolayers. Using both methods allows one to distinguish sampling artefactual features from real sample spectral features. In transflection experiments, amide I/II absorption bands down-shift 9/4 cm(-1), respectively, relative to the corresponding bands in transmission experiments. In all other spectral regions no pronounced frequency differences in spectral bands in transmission and transflection experiments were observed. Transmission and transflection infrared microscopy were used to obtain infrared spectra for unfixed and acetone- or formalin-fixed Vero cell monolayers. Formalin-fixed monolayers display spectra that are very similar to that obtained using unfixed cells. However, acetone fixation leads to considerable spectral modifications. For unfixed and formalin-fixed monolayers, a distinct band is observed at 1740 cm(-1). This band is absent in spectra obtained using acetone-fixed monolayers. The 1740 cm(-1) band is associated with cellular ester lipids. In support of this hypothesis, two bands at 2925 and 2854 cm(-1) are also found to disappear upon acetone fixation. These bands are associated with C--H modes of the cellular lipids. Acetone fixation also leads to modification of protein amide I and II absorption bands. This may be expected as acetone causes coagulation of soluble cellular proteins. Other spectral changes associated with acetone or formalin fixation in the 1400-800 cm(-1) region are discussed. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 921-930, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

On-line process monitoring and chemometric modeling with 2D fluorescence spectra obtained in recombinant E. coli fermentations

2D spectrofluorometry produces a large volume of spectral data during fermentation processes with recombinant E. coli, which can be analyzed using chemometric methods such as principal component analysis (PCA), principal component regression (PCR) and partial least square regression (PLS). An analysis of the spectral data by PCA results in scores and loadings that are not only visualized in the score-loading plots but are also used to monitor the fermentation processes on-line. The score plots provided useful qualitative information on four fermentation processes for the production of extracellular 5-aminolevulinic acid (ALA). Two chemometric models (PCR and PLS) were used to examine the correlation between the 2D fluorescence spectra and a few parameters of the fermentation processes. The results showed that PLS had slightly better calibration and prediction performance than PCR.     

Using FTIR to predict saccharification from enzymatic hydrolysis of alkali-pretreated biomasses

Fourier transform infrared, attenuated total reflectance (FTIR-ATR) spectroscopy combined with partial least squares (PLS) regression accurately predicted 72-h glucose and xylose conversions (g sugars/100 g potential sugars) and yields (g sugars/100 g dry solids) from cellulase-mediated hydrolysis of alkali-pretreated lignocellulose. Six plant biomasses that represent a variety of potential biofuel feedstocks--two switchgrass cultivars, big bluestem grass, a low-impact, high-diversity mixture of 32 species of prairie biomasses, mixed hardwood, and corn stover--were subjected to four levels of low-temperature NaOH pretreatment to produce 24 samples with a wide range of potential digestibility. PLS models were constructed by correlating FTIR spectra of pretreated samples to measured values of gluose and xylose conversions and yields. Variable selection, based on 90% confidence intervals of regression-coefficient matrices, improved the predictive ability of the models, while simplifying them considerably. Final models predicted sugar conversions with coefficient of determination for cross-validation (Q(2)) values of 0.90 for glucose and 0.89 for xylose, and sugar yields with Q(2) values of 0.92 for glucose and 0.91 for xylose. The sugar-yield models are noteworthy for their ability to predict enzymatic saccharification per mass dry solids without a priori knowledge of the composition of the solids. All peaks retained in the final regression coefficient matrices were previously assigned to chemical bonds and functional groups in lignocellulose, demonstrating that the models were based on real chemical information. This study demonstrates that FTIR spectroscopy combined with PLS regression can be used to rapidly estimate sugar conversions and yields from enzymatic hydrolysis of pretreated plant biomass.     

Analytical monitoring of alcoholic fermentation using NIR spectroscopy

Alcoholic fermentation under Saccharomyces cerevisiae yeasts is governed largely by glucose uptake, biomass formation, ethanol and glycerin production, and acidification. In this work, PLS calibration models were developed with a view to determining these analytical parameters from near infrared spectra and analytical data provided by the corresponding reference methods. The models were applied to a set of samples obtained from various fermentation processes. The glucose, ethanol, and biomass values predicted by the models exhibited a high correlation with those provided by the reference method.     

Effect of selective growth media on the differentiation of Salmonella enterica serovars by Fourier-Transform Mid-Infrared Spectroscopy

Salmonella enterica serovars are prevalent foodborne pathogens responsible for high numbers of salmonellosis each year. Complex Fourier-transform infrared (FTIR) spectra offer unique biochemical fingerprints of bacteria with bands due to major cellular components. Growth media effects on discrimination of Salmonella serovars by FTIR spectroscopy were investigated and a novel sample preparation technique was developed. S. enterica strains from six serovars were grown on xylose lysine desoxycholate (XLD), Miller-Mallinson (MM), and plate count (PCA) agar as a control (37 degrees C, 24 h). Isolated colonies were suspended in 50% acetonitrile and centrifuged; the remaining pellet was placed on an AMTIR (attenuated total reflectance) crystal and dried under vacuum. Classification models (Soft Independent Modeling of Class Analogy, SIMCA), generated from derivatized infrared spectra (1300-900 cm-1 or 1200-900 cm-1), successfully discriminated among Salmonella strains with major discrimination from 1000-970 cm-1 associated to stretching modes of O-specific polysaccharide chains of lipopolysaccharides. Sample treatment with acetonitrile enhanced safe handling of the bacteria, removed interfering signals and improved the discriminating ability of SIMCA. All media were able to discriminate the S. enterica strains studied, varying in discriminating peaks and class distances in SIMCA classification. This methodology, with the production of large libraries of pathogenic bacteria, could be applied for the rapid monitoring of bacterial contamination in food with minimal sample manipulation.     

Screening soy hydrolysates for the production of a recombinant therapeutic protein in commercial cell line by combined approach of near-infrared spectroscopy and chemometrics

Soy hydrolysates are widely used as the major nutrient sources for cell culture processes for industrial manufacturing of therapeutic recombinant proteins. The primary goal of this study was to develop a spectroscopy based chemometric method, a partial least squares (PLS), to screen soy hydrolysates for better yield of protein production (titers) in cell culture medium. Harvest titer values of 29 soy hydrolysate lots with production yield between 490 and 1,350 mg/L were obtained from shake flask models or from manufacture engineering runs. The soy hydrolysate samples were measured by near-infrared (NIR) in reflectance mode using an infrared fiber optic probe. The fiber optic probe could easily enable in situ measurement of the soy hydrolysates for convenient raw material screening. The best PLS calibration has a determination coefficient of R ²?=?0.887 utilizing no spectral preprocessing, the two spectral ranges of 10,000–5,376 cm^?1 and 4,980–4,484 cm^?1, and a rank of 6 factors. The cross-validation of the model resulted in a determination coefficient of R ²?=?0.741 between the predicted and actual titer values with an average standard deviation of 72 mg/L. Compared with the resource demanding shake flask model, the combination of NIR and chemometric modeling provides a convenient method for soy hydrolysate screening with the advantage of fast speed, low cost and non-destructive.     

Lipid quantification method using FTIR spectroscopy applied on cancer cell extracts

Reprogramming energy metabolism constitutes one of the hallmarks of cancer. Changes in lipid composition of cell membranes also appear early in carcinogenesis. Quantification of various molecules such as lipids evidences the modifications in the metabolism of tumour cells and can serve as potential markers for cancer diagnosis and treatment. Fourier Transform Infrared (FTIR) spectroscopy is a powerful tool used for the detection and characterization of various types of molecules. This technique remains an attractive approach as it is cheap (equipment and reagents), does not require high grade solvents or expensive internal standards, equipment is widely available in standard laboratories and the method is robust and suitable for routine analyses. In this work we established partial least square (PLS) models based on FTIR spectra able to quantify lipids in complex mixtures such as cell extracts. In the first part, we attempted to build PLS models with FTIR spectra of 53 mixtures of 8 well-characterized pure lipids. Second, the PLS models were verified using FTIR spectra of mixtures that did not contribute to the calibration. The third step was the validation of the models on lipid cell extracts. In order to obtain reference values for cell extracts, high performance liquid chromatography was carried out by AVANTI. The lipid distribution were globally similar with both techniques, PLS models and chromatography. Finally, the models were applied to determine the lipid composition of cells exposed to four treatments. We could not evidence significant changes in the lipid composition of cell extracts after treatment, in terms of polar head groups. However, the models established in this study appear reliable and could be applied for high throughput measurements. This article is part of a Special Issue entitled Tools to study lipid functions.     

食用调和油中花生油含量的近红外光谱分析

采用偏最小二乘法(PLS)等方法建立了食用调和油中花生油含量定量分析的近红外光谱定标模型。采集食用调和油样品在4 000 cm-1~10 000 cm-1范围内的近红外漫反射光谱,光谱经一阶导数处理后,采用偏最小二乘法建立样品中花生油含量的定标模型,并用Leave-one-out内部交叉验证法对模型进行验证。模型相关系数为0.99961,校正均方根RMSEC为0.830%。比较不同光谱预处理方法对定标模型的影响,结果表明一阶导数Corr.coeff最好。采用不同的化学计量学方法建立的定标模型中以偏最小二乘回归法最理想。     

Feasibility of Spectroscopic Characterization of Algal Lipids: Chemometric Correlation of NIR and FTIR Spectra with Exogenous Lipids in Algal Biomass

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.