Monitoring a complex medium fermentation with sample-sample two-dimensional FT-NIR correlation spectroscopy

The possibility of monitoring state transitions during the time course of a fermentation through the analysis of the on-line collected near-infrared (NIR) spectra with sample-sample two-dimensional correlation spectroscopy (SS-2DCoS) was investigated. SS-2DCoS has proved to be useful for extracting process information directly from the spectra collected on-line. The complexity of the system studied prevented the extraction of concentration profiles, but nevertheless, the application of SS-2DCoS enables the identification of fermentation state transitions due to metabolic and morphological changes of the microorganism.     

Rheology of filamentous fermentations

The performance of a bioreactor containing a filamentous fermentation broth is greatly influenced by the rheological properties of the broth. These properties are determined mainly by the concentration of biomass, its growth rate and morphology. Included in the morphology are such factors as the geometry of hyphae (length, diameter, branching frequency), hyphal flexibility and hyphal-hyphal interactions, which can all be affected by the operational design of the reactor. Thus, correlations describing viscosity as a function of biomass only are of limited value. A better understanding of the relations between morphology and rheology may be achieved by a combination of rheological and morphological studies.Rheological properties are normally determined using off-line measurements in-spite of associated problems with sample treatment influencing the results. Equipment for dynamic, on-line, measurement of morphology and rheology is available, but little used in filamentous fermentations. Controlling the rheological properties of mycelial fermentations may be difficult because of the great number of factors influencing mycelial development and/or hyphal-hyphal interactions.Polymer solutions are often used to simulate flow behaviour of filamentous fermentations and scale-up and mass transfer considerations are based on these studies. Although much information has been gained this way, the predictions developed do not include the effect of an active biomass on the mass transfer and flow properties of the culture. It is important to carry out studies on the non-homogeneous fermentation fluids, and develop correlations based on these studies.     

In-Line Monitoring of a High-Shear Granulation Process Using the Baseline Shift of Near Infrared Spectra

Although near infrared (NIR) spectra are primarily influenced by undesired variations, i.e., baseline shifts and non-linearity, and many applications of NIR spectroscopy to the real-time monitoring of wet granulation processes have been reported, the granulation mechanisms behind these variations have not been fully discussed. These variations of NIR spectra can be canceled out using appropriate pre-processing techniques prior to spectral analysis. The present study assessed the feasibility of directly using baseline shifts in NIR spectra to monitor granulation processes, because such shifts can reflect changes in the physical properties of the granular material, including particle size, shape, density, and refractive index. Specifically, OPUSGRAN^®, a novel granulation technology, was investigated by in-line NIR monitoring. NIR spectra were collected using a NIR diffuse reflectance fiber optic probe immersed in a high-shear granulator while simultaneously examining the morphology, particle size, density, strength, and Raman images of the mixture during granulation. The NIR baseline shift pattern was found to be characteristic of the OPUSGRAN^® technology and was attributed to variations in the light transmittance, reflection, and scattering resulting from changes in the physicochemical properties of the samples during granulation. The baseline shift also exhibited an inflection point around the completion of granulation, and therefore may be used to determine the endpoint of the process. These results suggest that a specific pattern of NIR baseline shifts are associated with the unique OPUSGRAN^® granulation mechanism and can be applied to monitor the manufacturing process and determine the endpoint.     

Effect of biomass concentration and mycelial morphology on fermentation broth rheology

The effect of biomass concentration and mycelial morphology on fungal fermentation broth rheological properties has been investigated. In previous work it had been shown that commonly used rheological parameters, such as the power law consistency and flow behavior indices, could be correlated successfully with the broth biomass concentration and clump morphological parameters of roughness and compactness. More recent work on a broader range of data showed a correlation between roughness and compactness; consequently, it was not correct to use both of these morphological variables simultaneously in rheological parameter correlations. Furthermore, earlier correlations were only made using clump morphological parameters, as clumps were found to be around 90% of the biomass in batch fermentations. In the present work it was found that the percentage of clumps fell to around 30% to 40% of a sample during the later stages of fed-batch fermentations. No clear relationship between the flow behavior index and biomass concentration was found, at least for those phases of the fermentation in which the viscosities were high enough for the rheology to be characterized by a disk turbine rheometer. The mean value of the flow behavior index was found to be 0.35 +/- 0.1 (standard deviation) throughout both batch and fed-batch fermentations, although some significant deviations from this value were observed early and very late in the fermentations. Correlations for the consistency index, measured using a disk turbine rheometer, were based on the biomass concentration and the mycelial size (represented by the mean projected area or the mean maximum dimension of all the mycelia). These correlations were reasonably successful for both fed-batch and batch fermentations. The correlation using the mean maximum dimension was preferred to that using the mean projected area, as the former is independent of magnification. The proposed correlation is: where K is the consistency index (Pa. s(n>)), C(m) is the biomass concentration as dry cell weight (g L(-1)), and D is the mean maximum dimension (microm). It should be noted that small changes in the exponent on the biomass concentration (alpha) may dramatically affect any predictions. Consequently, caution in the use of this correlation (and that based on mean projected area) is advocated, although its accuracy may be suitable for operational or design purposes.     

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.     

Assessment of near-infrared spectral information for rapid monitoring of bioprocess quality.

Access to real-time process information is desirable for consistent and efficient operation of bioprocesses. Near-infrared spectroscopy (NIRS) is known to have potential for providing real-time information on the quantitative levels of important bioprocess variables. However, given the fact that a typical NIR spectrum encompasses information regarding almost all the constituents of the sample matrix, there are few case studies that have investigated the spectral details for applications in bioprocess quality assessment or qualitative bioprocess monitoring. Such information would be invaluable in providing operator-level assistance on the progress of a bioprocess in industrial-scale productions. We investigated this aspect and report the results of our investigation. Near-infrared spectral information derived from scanning unprocessed culture fluid (broth) samples from a complex antibiotic production process was assessed for a data set that incorporated bioprocess variations. Principal component analysis was applied to the spectral data and the loadings and scores of the principal components studied. Changes in the spectral information that corresponded to variations in the bioprocess could be deciphered. Despite the complexity of the matrix, near-infrared spectra of the culture broth are shown to have valuable information that can be deconvoluted with the help of factor analysis techniques such as principal component analysis (PCA). Although complex to interpret, the loadings and score plots are shown to offer potential in process diagnosis that could be of value in the rapid assessment of process quality, and in data assessment prior to quantitative model development.     

Optimization of submerged culture process for the production of mycelial biomass and exo-polysaccharides by Cordyceps militaris C738

AIMS: The objective of the present study was to determine the optimal culture conditions for mycelial biomass and exo-polysaccharide (EPS) by Cordyceps militaris C738 in submerged culture. METHODS AND RESULTS: The optimal temperatures for mycelial biomass and EPS production were 20 degrees C and 25 degrees C, respectively, and corresponding optimal initial pHs were found to be 9 and 6, respectively. The suggested medium composition for EPS production was as follows: 6% (w/v) sucrose, 1% (w/v) polypeptone, and 0.05% (w/v) K2HPO4. The influence of pH on the fermentation broth rheology, morphology and EPS production of C. militaris C738 was carried out in a 5-l stirred-tank fermenter. The morphological properties were comparatively characterized by pellet roughness and compactness by use of image analyser between the culture conditions with and without pH control. The roughness and compactness of the pellets indicated higher values at pH-stat culture (pH 6.0), suggesting that larger and more compact pellets were desirable for polysaccharide production (0.91 g g(-1) cell d(-1). CONCLUSIONS: Under the optimized culture conditions (with pH control at 6), the maximum concentration of biomass and EPS were 12.7 g l(-1) and 7.3 g l(-1), respectively, in a 5-l stirred-tank fermenter. SIGNIFICANCE AND IMPACT OF THE STUDY: The critical effect of pH on fungal morphology and rheology presented in this study can be widely applied to other mushroom fermentation processes.     

A fractal model for the characterization of mycelial morphology

A new technique based on a fractal model has been developed for the quantification of the macroscopic morophology of mycelia. The morphological structuring is treated as a fractal object, and the fractal dimension, determined by an ultrasonic scattering procedure developed for the purpose, serves as a quantitative morphological index. Experimental observations reported earlier and simulations of mycelial growth, carried out using a probabilistic-geometric growth model developed for the purpose, both validate the applicability of the fractal model. In experiments with three different species, the fractal dimensions of pelletous structures were found to be in the range 1.45-2.0 and those of filamentous structures were in the range 1.9-2.7, with values around 2.0 representing mixed morphologies. Fractal dimensions calculated from simulated mycelia are in rough agreement with these ranges. The fractal dimension is also found to be relatively insensitive to the biomass concentration, as seen by dilution of the original broths. The relation between morphology and filtration properties of the broths has also been studied. The fractal dimension shows a strong correlation with the index of cake compressibility and with the Kozeny constant, two filtration parameters that are known to be morphology dependent. This technique could thus be used to develop correlations between the morphology, represented by the fractal dimension, and important morphology-dependent process variables. (c) 1993 John Wiley & Sons, Inc.     

Production of cephalosporin C in a fluidized-bed bioreactor

Production of cephalosporin C was investigated in a fluidized-bed bioreactor using bioparticles of Cephalosporium acremonium. Bioparticles were developed by forming a biofilm of growing hyphae around celite particles which contained spores of the microorganism. Production of the antibiotic was significantly improved by using bioparticles over the free mycelial culture, possibly due to the enhanced mass transfer capacity of the bioreactor system and successive generation of highly productive morphological forms of the microorganism. The maximum attainable titer of cephalosporin C from the bioreactor system was almost double that from a jar fermentor operation with a free mycelial culture of the same strain. The biofilm of the bioparticles became unstable as the fermentation proceeded. Morphological differentiation of the microorganism caused a gradual loss of biofilm and an increase of free cells in the culture broth. Additional feeding of a limited amount of methionine to the fermentation broth was not as effective as expected for improving the bioparticle stability. However, repeated use of the bioparticles revealed a strong possibility to improved the overall reactor performance since it allowed an enhanced production of the antibiotic with fewer free cells.     

PAT-Based Control of Fluid Bed Coating Process Using NIR Spectroscopy to Monitor the Cellulose Coating on Pharmaceutical Pellets

Current endeavor was aimed towards monitoring percent weight build-up during functional coating process on drug-layered pellets. Near-infrared (NIR) spectroscopy is an emerging process analytical technology (PAT) tool which was employed here within quality by design (QbD) framework. Samples were withdrawn after spraying every 15-Kg cellulosic coating material during Wurster coating process of drug-loaded pellets. NIR spectra of these samples were acquired using cup spinner assembly of Thermoscientific Antaris II, followed by multivariate analysis using partial least squares (PLS) calibration model. PLS model was built by selecting various absorption regions of NIR spectra for Ethyl cellulose, drug and correlating the absorption values with actual percent weight build up determined by HPLC. The spectral regions of 8971.04 to 8250.77 cm^?1, 7515.24 to 7108.33 cm^?1, and 5257.00 to 5098.87 cm^?1 were found to be specific to cellulose, where as the spectral region of 6004.45 to 5844.14 cm^?1was found to be specific to drug. The final model gave superb correlation co-efficient value of 0.9994 for calibration and 0.9984 for validation with low root mean square of error (RMSE) values of 0.147 for calibration and 0.371 for validation using 6 factors. The developed correlation between the NIR spectra and cellulose content is useful in precise at-line prediction of functional coat value and can be used for monitoring the Wurster coating process.     

NIR techniques create added values for the pellet and biofuel industry

A 2(3)-factorial experiment was carried out in an industrial plant producing biofuel pellets with sawdust as feedstock. The aim was to use on-line near infrared (NIR) spectra from sawdust for real time predictions of moisture content, blends of sawdust and energy consumption of the pellet press. The factors varied were: drying temperature and wood powder dryness in binary blends of sawdust from Norway spruce and Scots pine. The main results were excellent NIR calibration models for on-line prediction of moisture content and binary blends of sawdust from the two species, but also for the novel finding that the consumption of electrical energy per unit pelletized biomass can be predicted by NIR reflectance spectra from sawdust entering the pellet press. This power consumption model, explaining 91.0% of the variation, indicated that NIR data contained information of the compression and friction properties of the biomass feedstock. The moisture content model was validated using a running NIR calibration model in the pellet plant. It is shown that the adjusted prediction error was 0.41% moisture content for grinded sawdust dried to ca. 6-12% moisture content. Further, although used drying temperatures influenced NIR spectra the models for drying temperature resulted in low prediction accuracy. The results show that on-line NIR can be used as an important tool in the monitoring and control of the pelletizing process and that the use of NIR technique in fuel pellet production has possibilities to better meet customer specifications, and therefore create added production values.     

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.     

Dibutyryl c-AMP as an inducer of sporidia formation: Biochemical and antigenic changes during morphological differentiation of Karnal bunt (<Emphasis Type="Italic">Tilletia indica</Emphasis>) pathogen in axenic culture

Effect of dibutyryl adenosine 3′,5′-cyclic monophosphate (dbc-AMP), an analogue of c-AMP, was investigated on growth and morphological differentiation ofTilletia indica. Exponential growth was observed up to 21 days in both presence and absence of dbc-AMP; however, increasing concentration of dbc-AMP was deleterious to mycelial growth in liquid culture. A slow increase of mycelial biomass up to 21 days and decline at 30 days in the presence of 2.5 mM dbc-AMP was observed, therefore, this concentration was chosen in subsequent investigations. The inhibitory influence of dbc-AMP was further substantiated by decrease in soluble protein. The fungus on exposure to dbc-AMP experienced morphological differentiation from vegetative mycelial phase to sporogenous mycelial phase, and was induced to produce filiform sporidia. Use of quantitative ELISA further suggested that sporidia formation took more than 21 days in the presence of dbc-AMP. Variations of proteins during different stages ofT. indica grown in the presence and absence of dbc-AMP suggested the expression of stage-specific proteins or differential expression of proteins induced by dbc-AMP. The changes in expression of cell surface antigens as evidenced from decrease and increase binding of anti-mycelial and anti-sporidial antibodies in dbc-AMP treated culture by ELISA was further interpreted on the basis of morphological differentiation from mycelial to sporidial phase     

Fungal morphology and metabolite production in submerged mycelial processes

The use of fungi for the production of commercial products is ancient, but it has increased rapidly over the last 50 years. Fungi are morphologically complex organisms, differing in structure at different times in their life cycle, differing in form between surface and submerged growth, differing also with the nature of the growth medium and physical environment. Many genes and physiological mechanisms are involved in the process of morphogenesis. In submerged culture, a large number of factors contribute to the development of any particular morphological form. Factors affecting morphology include the type and concentration of carbon substrate, levels of nitrogen and phosphate, trace minerals, dissolved oxygen and carbon dioxide, pH and temperature. Physical factors affecting morphology include fermenter geometry, agitation systems, rheology and the culture modes, whether batch, fed-batch or continuous. In many cases, particular morphological forms achieve maximum performance. It is a very difficult task to deduce unequivocal general relationships between process variables, product formation and fungal morphology since too many parameters influence these interrelationships and the role of many of them is still not fully understood. The use of automatic image analysis systems during the last decade proved an invaluable tool for characterizing complex mycelial morphologies, physiological states and relationships between morphology and productivity. Quantified morphological information can be used to build morphologically structured models of predictive value. The mathematical modeling of the growth and process performance has led to improved design and operation of mycelial fermentations and has improved the ability of scientists to translate laboratory observations into commercial practice. However, it is still necessary to develop improved and new experimental techniques for understanding phenomena such as the mechanisms of mycelial fragmentation and non-destructive measurement of concentration profiles in mycelial aggregates. This would allow the establishment of a process control on a physiological basis. This review is focused on the factors influencing the fungal morphology and metabolite production in submerged culture.     

Spectrophotometric characteristics of the coccoid forms of actinomycetes]

IR spectra of thw whole cells of the coccoid forms (Mycococcus and Micrococcus) isolated from lithophilous lichen were compared with IR spectra of the collection cultures of Micrococcus and Arthrobacter. Generic spectral characteristics of Mycococcus and Micrococcus are presented. Spectral heterogeneity within the genus Arthrobacter complicates the diagnosis. The cultures of the Mycococcus genus were divided into three groups according to their spectral characteristics. Spectral scans of the studied coccoid forms differ from the scans of the mycelial actinomycetes, and their intensities within the range of 900-1200 cm-1 (lipids) decrease in the series Mycococcus, Arthrobacter, Micrococcus.     

Dependence of morphology on agitation intensity in fed-batch cultures of Aspergillus oryzae and its implications for recombinant protein production

We previously reported that, although agitation conditions strongly affected mycelial morphology, such changes did not lead to different levels of recombinant protein production in chemostat cultures of Aspergillus oryzae (Amanullah et al., 1999). To extend this finding to another set of operating conditions, fed-batch fermentations of A. oryzae were conducted at biomass concentrations up to 34 g dry cell weight/L and three agitation speeds (525, 675, and 825 rpm) to give specific power inputs between 1 and 5 kWm(-3). Gas blending was used to control the dissolved oxygen level at 50% of air saturation except at the lowest speed where it fell below 40% after 60-65 h. The effects of agitation intensity on growth, mycelial morphology, hyphal tip activity, and recombinant protein (amyloglucosidase) production in fed-batch cultures were investigated. In the batch phase of the fermentations, biomass concentration, and AMG secretion increased with increasing agitation intensity. If in a run, dissolved oxygen fell below approximately 40% because of inadequate oxygen transfer associated with enhanced viscosity, AMG production ceased. As with the chemostat cultures, even though mycelial morphology was significantly affected by changes in agitation intensity, enzyme titers (AGU/L) under conditions of substrate limited growth and controlled dissolved oxygen of >50% did not follow these changes. Although the measurement of active tips within mycelial clumps was not considered, a dependency of the specific AMG productivity (AGU/g biomass/h) on the percentage of extending tips was found, suggesting that protein secretion may be a bottle-neck in this strain during fed-batch fermentations.     

Identification of optimal hyperspectral bands for estimation of rice biophysical parameters

The present study aims to identify the narrow spectral bands that are most suitable for characterizing rice biophysical parameters. The data used for this study come from ground-level hyperspectral reflectance measurements for five rice species at three levels of nitrogen fertilization during the growing period. Reflectance was measured in discrete narrow bands between 350 and 2 500 nm. Observed rice biophysical parameters included leaf area index （LAI）, wet biomass and dry biomass. The stepwise regression method was applied to identify the optimal bands for rice biophysical parameter estimation. This research indicated that combinations of four narrow bands in stepwise regression models explained 69% to 83% variability for LAI, 56% to 73% for aboveground wet biomass and 70% to 83% for leaf wet biomass. An overwhelming proportion of rice information was in a particular portion of near infrared （NIR） （1 100-1 150 nm）, red-edge （700-750 nm）, and a longer portion of green （550-600 nm）. These were followed by the moisture-sensitive NIR （950-1 000 nm）, the intermediate portion of shortwave infrared （SWlR） （1 650-1 700 nm）, and another portion of NIR （1 000-1 050 nm）.