Determination of solid-state fungal growth by Fourier transform infrared-photoacoustic spectroscopy

Abstract Photoacoustic spectroscopy (PAS) does not require optically transparent samples and is, therefore, well suited for analysis of solid-state samples. Fourier transform infrared (FTIR)-PAS of solid materials containing protein exhibited strong absorption in the amide I and amide II regions of the IR spectrum. Growth of a filamentous fungus, Phanerochaete chrysosporium , on cellulose discs was quantitatively determined by monitoring amide I absorption with FTIR-PAS.     

Quantitation of aliphatic suberin in Quercus suber L. cork by FTIR spectroscopy and solid-state (13)C-NMR spectroscopy

This work determined that the percentage of suberin in cork may be found by solid-state (13)C cross polarization/magic angle spinning (CP/MAS) NMR spectroscopy and by FTIR with photoacoustic detection (FTIR-PAS) spectroscopy. A linear relationship is found between the suberin content measured through CP/MAS spectral areas and that measured gravimetrically. Furthermore, application of a partial least squares (PLS1) regression model to the NMR and gravimetric data sets clearly correlates the two sets, enabling suberin quantification with 90% precision. Suberin quantitation by FTIR-PAS spectroscopy is also achieved by a PLS1 regression model, giving 90% accurate estimates of the percentage of suberin in cork. Therefore, (13)C-CP/MAS NMR and FTIR-PAS proved to be useful and accurate noninvasive techniques to quantify suberin in cork, thus avoiding the traditional time consuming and destructive chemical methods.     

On-line biomass measurements in bioreactor cultivations: comparison study of two on-line probes

Two on-line probes for biomass measurement in bioreactor cultivations were evaluated. One probe is based on near infrared (NIR) light absorption and the other on dielectric spectroscopy. The probes were used to monitor biomass production in cultivations of several different microorganisms. Differences in NIR probe response compared to off-line measurement methods revealed that the most significant factor affecting the response was cell shape. The NIR light absorption method is more developed and reliable for on-line in situ biomass estimation than dielectric spectroscopy. The NIR light absorption method is, however, of no significant use, when the cultivation medium is not clear, and especially in processes using adsorbents or solid matrix for the microorganism to grow on. The possibilities offered by dielectric spectroscopy are impressive, but the on-line probe technology needs to be improved.     

Computer-assisted analysis of photoacoustic spectra of solid and solution state cyanide methemoglobin

A computer-assisted method for analyzing photoacoustic spectra has been developed. Using this analysis, the relative absorption spectrum and either the chromophore concentration or thermal diffusivity characteristic of a sample can be derived from its photoacoustic spectrum. We have demonstrated the accuracy of the method by analyzing photoacoustic spectra of solution and crystalline-phase bovine cyanide methemoglobin. BASIC and FORTRAN routines used to collect and to analyze photoacoustic spectra are described. Photoacoustic spectroscopy can be used in conjunction with the analytical method presented here to obtain accurate absorption spectra from a variety of solid, opaque, and/or turbid samples.     

Organic and inorganic carbon in paddy soil as evaluated by mid-infrared photoacoustic spectroscopy

Paddy soils are classified as wetlands which play a vital role in climatic change and food production. Soil carbon (C), especially soil organic C (SOC), in paddy soils has been received considerable attention as of recent. However, considerably less attention has been given to soil inorganic carbon (SIC) in paddy soils and the relationship between SOC and SIC at interface between soil and the atmosphere. The objective of this research was to investigate the utility of applying Fourier transform mid-infrared photoacoustic spectroscopy (FTIR-PAS) to explore SOC and SIC present near the surface (0-10 μm) of paddy soils. The FTIR-PAS spectra revealed an unique absorption region in the wavenumber range of 1,350-1,500 cm(-1) that was dominated by C-O (carbonate) and C-H bending vibrations (organic materials), and these vibrations were used to represented SIC and SOC, respectively. A circular distribution between SIC and SOC on the surface of paddy soils was determined using principal component analysis (PCA), and the distribution showed no significant relationship with the age of paddy soil. However, SIC and SOC were negatively correlated, and higher SIC content was observed near the soil surface. This relationship suggests that SIC in soil surface plays important roles in the soil C dynamics.     

Variability of cork from Portuguese Quercus suber studied by solid-state (13)C-NMR and FTIR spectroscopies.

A new approach is presented for the study of the variability of Portuguese reproduction cork using solid-state (13)C-NMR spectroscopy and photoacoustic (PAS) FTIR (FTIR-PAS) spectroscopy combined with chemometrics. Cork samples were collected from 12 different geographical sites, and their (13)C-cross-polarization with magic angle spinning (CP/MAS) and FTIR spectra were registered. A large spectral variability among the cork samples was detected by principal component analysis and found to relate to the suberin and carbohydrate contents. This variability was independent of the sample geographical origin but significantly dependent on the cork quality, thus enabling the distinction of cork samples according to the latter property. The suberin content of the cork samples was predicted using multivariate regression models based on the (13)C-NMR and FTIR spectra of the samples as reported previously. Finally, the relationship between the variability of the (13)C-CP/MAS spectra with that of the FTIR-PAS spectra was studied by outer product analysis. This type of multivariate analysis enabled a clear correlation to be established between the peaks assigned to suberin and carbohydrate in the FTIR spectrum and those appearing in the (13)C-CP/MAS spectra.     

Characterization of lignin in situ by photoacoustic spectroscopy

Photoacoustic spectroscopy is a recently developed nondestructive analytical technique that provides ultraviolet, visible, and infrared absorption spectra from intensely light scattering, solid, and/or optically opaque materials not suitable for conventional spectrophotometric analysis. In wood and other lignocellulosics, the principal ultraviolet absorption bands, in the absence of photosynthetic pigments, arise from the aromatic lignin component of the cell walls. Photoacoustic spectra of extracted lignin fragments (milled wood lignin) and synthetic lignin-like polymers contain a single major absorption band at 280 nanometers with an absorption tail extending beyond 400 nanometers. Photoacoustic spectra of pine, maple, and oak lignin in situ contain a broad primary absorption band at 300 nanometers and a longer wavelength shoulder around 370 nanometers. Wheat lignin in situ, on the other hand, exhibits two principle absorption peaks, at 280 nanometers and 320 nanometers. The presence of absorption bands at wavelengths greater than 300 nanometers in intact lignin could result from (a) interacting, nonconjugated chromophores, or (b) the presence of more highly conjugated structural components formed as the result of oxidation of the polymer. Evidence for the latter comes from the observation that, on the outer surface of senescent, field-dried wheat culms (stems), new absorption bands in the 350 to 400 nanometer region predominate. These new bands are less apparent on the outer surface of presenescent wheat culms and are virtually absent on the inner surface of either senescent or presenescent culms, suggesting that the appearance of longer wavelength absorption bands in senescent wheat is the result of accumulated photochemical modifications of the ligin polymer. These studies also demonstrate photoacoustic spectroscopy to be an important new tool for the investigation of insoluble plant components.     

Enzymatic isolation and structural characterisation of polymeric suberin of cork from Quercus suber L

An enzymatic method has been used to isolate, for the first time, polymeric suberin from the bark of Quercus suber L. or cork. This was achieved by solvent extraction (dichloromethane, ethanol and water), followed by a step-by-step enzymatic treatment with cellulase, hemicellulase and pectinase, and a final extraction with dioxane/water. The progress of suberin isolation was monitored by Fourier transform infrared spectroscopy using a photoacoustic cell (FTIR-PAS). The material obtained (polymeric suberin (PS)) was characterised by solid-state and liquid-state nuclear magnetic resonance, FTIR-PAS and vapour pressure osmometry, and compared with the suberin fraction obtained by alkaline depolymerisation (depolymerised suberin (DS)). The results showed that PS is an aliphatic polyester of saturated and unsaturated fatty acids, with an average molecular weight (M(w)) of 2050 g mol(-1). Although this fraction represents only 10% of the whole suberin of cork, its polymeric nature gives valuable information about the native form of the polymer. DS was found to have an average M(w) of 750 g mol(-1) and to comprise a significant amount of acidic and alcoholic short aliphatic chains.     

用光声光谱技术测量鼻咽癌细胞的光谱吸收特性

为了解人鼻咽癌细胞的光谱吸收特性,用双光束归一化光声光谱技术测量三株人鼻咽癌细胞(SUNE-1,CNE-2,CNE-1)的归一化吸收光谱和光谱吸收系数.结果表明(1)在波长4000～6000A范围内,这三株人鼻咽癌细胞的光谱吸收系数大小为SUNE-1＞CNE-2＞CNE-1;(2)在波长4200A处平均相对光声信号值为SUNE-11.36,CNE-21.20,CNE-11.10,三者之间的差异具有显著性(P值＝0.012).人鼻咽癌细胞株SUNE-1,CNE-2和CNE-1具有不同的光声光谱特性.     

植物叶片的相位解析光声光谱和光声相位谱

用相位解析法获得的未损伤植物叶片叶肉组织的光声光谱,与叶片的光声相位谱进行了比较．发现植物叶片的光声相位谱与叶绿体光吸收带之间呈现互补关系．叶片表皮的光声相位谱存在持有的波谷结构．不同调制频率的光声相位谱有较大的差异．这些现象表明光声相位谱与光声光谱一样,也可对未损伤的层状生物样品作深度剖面分析．     

Variation in Venoms of <Emphasis Type="Italic">Polybia Paulista</Emphasis> Von Ihering and <Emphasis Type="Italic">Polybia Occidentalis</Emphasis> Olivier (Hymenoptera: Vespidae), Assessed by the FTIR-PAS Technique

Wasps are able to synthesize toxic compounds known as venoms, which form a part of a mechanism to overcome prey and also to defend their colonies. Study of the compounds that constitute these substances is essential in order to understand how this defense mechanism evolved, since there is evidence that the venoms can vary both intra- and interspecifically. Some studies have used liquid and gas chromatography as a reliable technique to analyze these compounds. However, the use of Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) to analyze the variations in venom’s chemical profile has been proposed recently. This study evaluated whether the FTIR-PAS technique is effective for assessing the role of environmental factors on intra- and interspecific differences in the venom of the wasps Polybia paulista Von Ihering and Polybia occidentalis Olivier by FTIR-PAS. The colonies were collected in three municipalities of Mato Grosso do Sul, Brazil, in different types of environments. The results showed that the venoms of P. paulista and P. occidentalis differed significantly in profile. In addition, the intraspecific differences in the venom’s chemical profile of P. paulista are related to the type of environment where they nested, regardless of the geographical distance between the nests. The FTIR-PAS technique proved to be reliable and effective to evaluate the variations in the venom’s chemical profile in social wasps.     

Growth kinetics of microorganisms with various ecological strategies in a dialysis culture at low specific growth rates

A dialysis culture was found to be most suitable for studying the metabolism of microorganisms at a low specific growth rate. The biomass of all microorganisms studied in the dialysis culture increased with time in a linear fashion; hence, the energy spent for growth decreased in proportion to a decrease in the specific growth rate. Microorganisms growing in oligotrophous environment (Arthrobacter globiformis and Lipomyces tetrasporus ) spent much less energy comparing to microorganisms from eutrophic habitats (Pseudomonas fluorescens and Debaryomyces formicarius ).     

A new biophysics approach using photoacoustic spectroscopy to study the DNA-ethidium bromide interaction

We have examined the binding processes of ethidium bromide interacting with calf thymus DNA using photoacoustic spectroscopy. These binding processes are generally investigated by a combination of absorption or fluorescence spectroscopies with hydrodynamic techniques. The employment of photoacoustic spectroscopy for the DNA-ethidium bromide system identified two binding manners for the dye. The presence of two isosbestic points (522 and 498 nm) during DNA titration was evidence of these binding modes. Analysis of the photoacoustic amplitude signal data was performed using the McGhee-von Hippel excluded site model. The binding constant obtained was 3.4 x 10(8) M(bp)(-1), and the number of base pairs excluded to another dye molecule by each bound dye molecule (n) was 2. A DNA drug dissociation process was applied using sodium dodecyl sulfate to elucidate the existence of a second and weaker binding mode. The dissociation constant determined was 0.43 mM, whose inverse value was less than the previously obtained binding constant, demonstrating the existence of the weaker binding mode. The calculated binding constant was adjusted by considering the dissociation constant and its new value was 1.2 x 10(9) M(bp)(-1) and the number of excluded sites was 2.6. Using the photoacoustic technique it is also possible to obtain results regarding the dependence of the quantum yield of the dye on its binding mode. While intercalated between two adjacent base pairs the quantum yield found was 0.87 and when associated with an external site it was 0.04. These results reinforce the presence of these two binding processes and show that photoacoustic spectroscopy is more extensive than commonly applied spectroscopies.     

Biosynthesis of protein by microscopic fungi in solid state fermentation. I. Selection of Aspergillus strain for enrichment of starchy raw materials in protein

A fungal strain suitable for protein enrichment of starchy raw materials was selected by evaluation of the growth rate, results of protein biosynthesis in solid state fermentation (SSF) and assessment of fungal biomass. The strain Aspergillus oryzae A.or. 11 selected for further studies was characterized by the radial growth rate K_r of about 300 μm/h and by the specific growth rate μ of about 0.100 h^?1. Fungal biomass contained about 32% of crude protein in dry matter. The digestibility of this protein in vitro was close to 75%. Protein analysis for amino acids showed that the content of exogenous amino acids approached that in protein of the FAO standard. As a result of 34 — h culture of this strain in solid medium, net protein increased by about 5.0 g/100 g of starting medium d.m. at the cost of decomposition of about 20 g carbohydrates.     

A new combined differential-discrete cellular automaton approach for biofilm modeling: application for growth in gel beads

The theoretical basis and quantitative evaluation of a new approach for modeling biofilm growth are presented here. Soluble components (e.g., substrates) are represented in a continuous field, whereas discrete mapping is used for solid components (e.g., biomass). The spatial distribution of substrate is calculated by applying relaxation methods to the reaction-diffusion mass balance. A biomass density map is determined from direct integration in each grid cell of a substrate-limited growth equation. Spreading and distribution of biomass is modeled by a discrete cellular automaton algorithm. The ability of this model to represent diffusion-reaction-microbial growth systems was tested for a well-characterized system: immobilized cells growing in spherical gel beads. Good quantitative agreement with data for global oxygen consumption rate was found. The calculated concentration profiles of substrate and biomass in gel beads corresponded to those measured. Moreover, it was possible, using the discrete spreading algorithm, to predict the spatial two- and three-dimensional distribution of microorganisms in relation to, for example, substrate flux and inoculation density. The new technique looks promising for modeling diffusion-reaction-microbial growth processes in heterogeneous systems as they occur in biofilms.     

Structural studies of phosvitin in solution and in the solid state

Phosvitin, a highly phosphorylated glycoprotein, represents the major fraction of hen egg yolk phosphoproteins. Circular dichroism, Fourier transform infrared spectroscopy, and Fourier transform infrared photoacoustic and fluorescence spectroscopic methods were employed to determine the secondary structure of the protein in both the solid and solution phases. This was supplemented by a Chou-Fasman type of predictive algorithm for the first 25 residues at the N terminus of the dephosphorylated protein. A three-compartment model consisting of alpha-helical, beta-sheet, and beta-turn components with beta-turns occurring at the interface between alpha-helical and beta-sheet regions in the proximity of O-phosphoserine residues is suggested from the combined analyses. Beta-sheets appear to be the dominant secondary structural component in phosvitin in the solid and solution phases. The suggested model bears many similarities to other phosphoproteins reported in the literature. The secondary structure of phosvitin is observed to be sensitive to environmental factors as previously reported although the present studies differ in some respects from earlier results. Preliminary results suggest that Ca2+ ions trigger a decrease in beta-sheet structure at pH 2.     

Integrating photoacoustic microscopy with other imaging technologies for multimodal imaging

As a hybrid optical microscopic imaging technology, photoacoustic microscopy images the optical absorption contrasts and takes advantage of low acoustic scattering of biological tissues to achieve high-resolution anatomical and functional imaging. When combined with other imaging modalities, photoacoustic microscopy-based multimodal technologies can provide complementary contrast mechanisms to reveal complementary information of biological tissues. To achieve intrinsically and precisely registered images in a multimodal photoacoustic microscopy imaging system, either the ultrasonic transducer or the light source can be shared among the different imaging modalities. These technologies are the major focus of this minireview. It also covered the progress of the recently developed penta-modal photoacoustic microscopy imaging system featuring a novel dynamic focusing technique enabled by OCT contour scan.     

Comparison of solid and solution state protein structures. Photoacoustic study of solid state bovine methemoglobin derivatives

In an effort to examine the consequences of phase state changes on protein structures, optical absorption spectra of several bovine hemoglobin derivatives in lyophilized, ammonium sulfate-precipitated, and crystalline states were examined. Absorption spectra were used to compare protein conformations. Differences between solution and several solid state spectra were apparent. They were greatest for lyophilized preparations, were inhibited by ligand binding, the tightest binding ligand being the most effective inhibitor, and were substantially or totally reversible upon dissolving the solid state protein. Though the magnitude of spectral changes varied, they all indicated characteristically different proportions of the protein were converted to a reversible hemichrome in each type of solid state preparation. The extent of hemichrome formation was correlated with the degree to which the protein tends to be dehydrated in each solid state preparation. This may reflect the role of water in determining the three-dimensional structure of hemoglobin. Absorption spectra of solid state hemoglobin preparations were obtained from photoacoustic spectra using a novel method for spectral analysis. Results presented here demonstrate the utility of this procedure in probing structures of solid state proteins.     

In situ optical spectroscopy of some systems of biological interest

Monitoring the optical absorption or emission spectrum of a condensed phase sample offers information about the supramolecular assembly, packing effects and other features characteristic of the phase that would be missed when one studies solution-state spectra. We have used the technique of photoacoustic spectroscopy to study intact biological specimens, such as algae, parasite cells and the eye lens. Such a study has offered information about the status of endogenous hemin inPlasmodium cells and the mode of interaction of antimalarial drugs of the chloroquine class therein. We have also attempted to doin situ fluorescence spectroscopy on isolated intact eye lenses, which has enabled us to follow the photochemistry and the status of the photoproduct of the oxidation of the trp residues of the crystallins of the lens.     

Screening of poly-β-hydroxybutyrate-producing microorganisms using Fourier transform infrared spectroscopy

An alternative method for the analysis of poly--hydroxybutyrate (PHB) in microbial biomass based on FTIR spectroscopy was developed in which a semi-transparent, thin pellet of biomass was made with KBr and scanned by IR spectroscopy, from 400 cm^–1to 2000 cm^–1. The IR spectra of the target biomass showed a distinct absorbance band of the carboxylic ester group at 1724.03 cm^–1. The technique can be used to screen large numbers of microorganisms for PHB production.