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Exopolysaccharide (EPS) production by Ganoderma lucidum BCCM 31549 immobilised on polyurethane foam (PUF) cubes exhibited high ability for continuous EPS production in a repeated-batch fermentation (RBF). RBF by immobilised mycelium was optimised for broth replacement time point and broth replacement ratio. Interestingly, seven batches could be fermented consecutively in the shake flask for 55 days using 80% broth ratio and at day 13 time point, where this smart immobilised approach was deployed. Fermentation time was reduced from 13 days to 6 days, and the medium usage was also reduced due to the broth replacement volume procedure. Critically, immobilised G. lucidum mycelium grown on PUF cubes could be reused at least for seven successive cycles without any loss of EPS efficiencies with the PUF cubes retained the original structure, pointing out to be an ideal immobilisation matrix. The EPS productivity in immobilised culture at 0.045 g/L day−1 was higher than the value in a freely suspended culture (0.029 g/L day−1). The proposed immobilisation strategy proved that PUF as a physical carrier would provide an economical solution to enhance EPS production for extended liquid fermentation of mushrooms.

Exopolysaccharide (EPS) productivity of the slow-growing Ganoderma lucidum BCCM 31549 mushroom was enhanced in repeated batch fermentation (RBF) process by reducing the fermentation time. The results showed that by using 2.5-L STR unbaffled bioreactor, the fermentation time could be reduced from 10 days per batch to 5 days, due to lag-phase elimination by adopting a RBF strategy. RBF approach was also shown to achieve a semi-continuous mode of production by eliminating the inoculation time between each cycle. The fermentation period was shortened from 50 to 25 days in five consecutive cycles of RBF, with the productivity of EPS increased from 0.81 g/L day−1 to 1.32 g/L day−1 by using N-limiting medium. The presence of clamp connections in RBF ensures the reproducibility of this strategy. The proposed strategy proved to provide easy operation and thus can be adapted for EPS production.

Corynebacterium glutamicum (C. glutamicum) is a highly promising alternative prokaryotic host for recombinant protein expression, as it possesses several significant advantages over Escherichia coli (E. coli), the currently leading bacterial protein expression system. During the past decades, several experimental techniques and vector components for genetic manipulation of C. glutamicum have been developed and validated, including strong promoters for tightly regulating target gene expression, various types of plasmid vectors, protein secretion systems and methods of genetically modifying the host strain genome to improve protein production potential. This review critically discusses current progress in establishing C. glutamicum as a host for recombinant protein expression, and examines, in depth, some successful case studies of actual application of this expression system. The established "expression tool box" for developing novel constructs based on C. glutamicum as a host are also evaluated. Finally, the existing issues and solutions in process development with C. glutamicum as a host are specifically addressed.

SeaBioTech is an EU-FP7 project designed and driven by SMEs to create innovative marine biodiscovery pipelines as a means to convert the potential of marine biotechnology into novel industrial products for the pharmaceutical, cosmetic, aquaculture, functional food and industrial chemistry sectors. To achieve its goals, SeaBioTech brings together leading experts in biology, genomics, natural product chemistry, bioactivity testing, industrial bioprocessing, legal aspects, market analysis and knowledge exchange. SeaBioTech targets novel marine endosymbiotic bacteria from unique and previously untapped habitats, including geothermal intertidal biotopes in Iceland, hydrothermal vent fields and deep-sea oligotrophic basins of the Eastern Mediterranean Sea and underexplored areas of Scottish coasts that are likely to be highly productive sources of new bioactive compounds. This chapter describes the 4 years of activity in the SeaBioTech project, which resulted in a robust, validated workflow suitable for evaluating unexplored activities in marine samples to prioritize potential products for a biotechnological pipeline. An improved integrated methodology involving metagenomics and metabolomics was extensively utilized to prioritize five extremophiles as potential antibiotics, anticancer drugs and novel drugs against metabolic diseases as well as new pharmaceutical excipients to the pipeline. A centralized biobank repository, which included a database of information, was established for future bioprospecting activities. For future marine bioprospecting activities, a harmonized legal position was put together in collaboration with other EU-FP7 blue biotechnology projects.

Prediction of soluble protein expression levels in Escherichia coli based on the nature of protein itself remains a challenge for bioprocess development (BD). This review will critically discuss the current efforts and achievements that employ computational approaches to develop prediction models for soluble protein expression in E. coli. The contrast between the remarkable progresses made on the predictive models achieved by bioinformatics and their relatively infrequent application in BD will be explained. The effects of process-relevant variables at four different levels on the expression of heterologous proteins, for example, gene, vector, host cell and cultivation process, and also a critical comparison of several established bioinformatics tools for predicting expression levels will be presented. The potential utility of this emergent technology to increase the efficiency of BD strategies and thereby to reduce the cost of establishing a process for soluble protein expression are critically examined.

Ganoderma lucidum BCCM 31549 has a long established role for its therapeutic activities. In this context, much interest has focused on the possible functions of the (1,3)-β-D-glucan (G) produced by these cultures in a stirred-tank bioreactor and extracted from their underutilized mycelium. In the existing study, we report on the systematic production of G, and its sulfated derivative (GS). The aim of this study was to investigate G and its GS from G. lucidum in terms of their antibacterial properties and cytotoxicity spectrum against human prostate cells (PN2TA) and human caucasian histiocytic lymphoma cells (U937). (1)H NMR for both G and GS compounds showed β-glycosidic linkages and structural similarities when compared with two standards (laminarin and fucoidan). The existence of characteristic absorptions at 1,170 and 867 cm(-1) in the FTIR (Fourier Transform Infrared Spectroscopy) for GS demonstrated the successful sulfation of G. Only GS exhibited antimicrobial activity against a varied range of test bacteria of relevance to foodstuffs and human health. Moreover, both G and GS did not show any cytotoxic effects on PN2TA cells, thus helping demonstrate the safety of these polymers. Moreover, GS showed 40% antiproliferation against cancerous U937 cells at the low concentration (60 μg/ ml) applied in this study compared with G (10%). Together, this demonstrates that sulfation clearly improved the solubility and therapeutic activities of G. The water-soluble GS demonstrates the potential multifunctional effects of these materials in foodstuffs.

Large-scale commercial bioprocesses that manufacture biopharmaceutical products such as monoclonal antibodies generally involve multiple bioreactors operated in parallel. Spectra recorded during in situ monitoring of multiple bioreactors by multiplexed fiber-optic spectroscopies contain not only spectral information of the chemical constituents but also contributions resulting from differences in the optical properties of the probes. Spectra with variations induced by probe differences cannot be efficiently modeled by the commonly used multivariate linear calibration models or effectively removed by popular empirical preprocessing methods. In this study, for the first time, a calibration model is proposed for the analysis of complex spectral data sets arising from multiplexed probes. In the proposed calibration model, the spectral variations introduced by probe differences are explicitly modeled by introducing a multiplicative parameter for each optical probe, and then their detrimental effects are effectively mitigated through a "dual calibration" strategy. The performance of the proposed multiplex calibration model has been tested on two multiplexed spectral data sets (i.e., MIR data of ternary mixtures and NIR data of bioprocesses). Experimental results suggest that the proposed calibration model can effectively mitigate the detrimental effects of probe differences and hence provide much more accurate predictions than commonly used multivariate linear calibration models (such as PLS) with and without empirical data preprocessing methods such as orthogonal signal correction, standard normal variate, or multiplicative signal correction.

The effects of oxygen availability and induction culture biomass upon production of an industrially important monoamine oxidase (MAO) were investigated in fed-batch cultures of a recombinant E. coli. For each induction cell biomass 2 different oxygenation methods were used, aeration and oxygen enriched air. Induction at higher biomass levels increased the culture demand for oxygen, leading to fermentative metabolism and accumulation of high levels of acetate in the aerated cultures. Paradoxically, despite an almost eight fold increase in acetate accumulation to levels widely reported to be highly detrimental to protein production, when induction wet cell weight (WCW) rose from 100% to 137.5%, MAO specific activity in these aerated processes showed a 3 fold increase. By contrast, for oxygenated cultures induced at WCW's 100% and 137.5% specific activity levels were broadly similar, but fell rapidly after the maxima were reached. Induction at high biomass levels (WCW 175%) led to very low levels of specific MAO activity relative to induction at lower WCW's in both aerated and oxygenated cultures. Oxygen enrichment of these cultures was a useful strategy for boosting specific growth rates, but did not have positive effects upon specific enzyme activity. Based upon our findings, consideration of the amino acid composition of MAO and previous studies on related enzymes, we propose that this effect is due to oxidative damage to the MAO enzyme itself during these highly aerobic processes. Thus, the optimal process for MAO production is aerated, not oxygenated, and induced at moderate cell density, and clearly represents a compromise between oxygen supply effects on specific growth rate/induction cell density, acetate accumulation, and high specific MAO activity. This work shows that the negative effects of oxygen previously reported in free enzyme preparations, are not limited to these acellular environments but are also discernible in the sheltered environment of the cytosol of E. coli cells.

There is considerable interest in exploiting the novel physical and biological properties of microbial exopolysaccharides in industry and medicine. For economic and scientific reasons, large scale production under carefully monitored and controlled conditions is required. Producing exopolysaccharides in industrial fermenters poses several complex bioengineering and microbiological challenges relating primarily to the very high viscosities of such culture media, which are often exacerbated by the producing organism's morphology. What these problems are, and the strategies for dealing with them are discussed critically in this review, using pullulan, curdlan, xanthan, and fungal β-glucans as examples of industrially produced microbial exopolysaccharides. The role of fermenter configuration in their production is also examined.

Fungi are amongst the most industrially important microorganisms in current use within the biotechnology industry. Most such fungal cultures are highly aerobic in nature, a character that has been frequently referred to in both reactor design and fungal physiology. The most fundamentally significant outcome of the highly aerobic growth environment in fermenter vessels is the need for the fungal culture to effectively combat in the intracellular environment the negative consequences of high oxygen transfer rates. The use of oxygen as the respiratory substrate is frequently reported to lead to the development of oxidative stress, mainly due to oxygen-derived free radicals, which are collectively termed as reactive oxygen species (ROS). Recently, there has been extensive research on the occurrence, extent, and consequences of oxidative stress in microorganisms, and the underlying mechanisms through which cells prevent and repair the damage caused by ROS. In the present study, we critically review the current understanding of oxidative stress events in industrially relevant fungi. The review first describes the current state of knowledge of ROS concisely, and then the various antioxidant strategies employed by fungal cells to counteract the deleterious effects, together with their implications in fungal bioprocessing are also discussed. Finally, some recommendations for further research are made.

Thermophilic aerobic digestion (TAD) of heteroxylan waste was implemented at waste load of 30gL(-1) with mineral nitrogen supplementation to study effect of the process on waste degradation, protein accretion and quality. Digestions were carried out at 45 50, 55, 60 and 65 degrees C using Bacillusstearothermophilus in a CSTR under batch conditions at 1.0vvm aeration rate, pH 7.0 for a maximum of 120h. Amylase and xylanase activities appeared rapidly in the digest, while basal protease activity appeared early in the digestion and increased towards end of the processes. Highest degradation of volatile suspended solid, hemicellulose and fibre occurred at 55 degrees C while highest degradation of total suspended solid occurred at 60 degrees C. Highest protein accretion (258.8%) and assimilation of mineral nitrogen and soluble protein occurred at 55 degrees C. The % content of amino acids of digest crude protein increased relative to raw waste and with digestion temperature. Quality of digest protein was comparable to the FAO standard for feed use. TAD has potentials for use in the protein enrichment of waste.

Filamentous fungi are industrially important organisms because of their ability to produce secondary metabolites, and as expression systems for heterologous proteins. Since secretion of secondary metabolites in filamentous fungi is associated with the older, more vacuolated parts of the hyphae culture morphology is a crucial reflection of their physiology in submerged cultures. Therefore, any factor affecting fungal morphology will in turn affect secondary metabolite production. In this study we examine and report the effects of elevated levels of the potent morphogen, carbon dioxide upon morphology and antibiotic production in an industrially important fungal system. The effects of elevated CO2 levels on cephalosporin C (CPC) production, metabolic performance and morphological changes in batch cultures of Acremonium chrysogenum (ATCC 48272) were investigated. High levels of dCO2 led to reduction of CPC production by up to 36%. Both metabolic activity and substrate uptake were severely inhibited by elevated CO2, especially at 15 and 20% CO2. Both micro and macromorphology of A. chrysogenum were profoundly changed in bioprocesses sparged with CO2 especially at high levels, and these changes were a contributory cause of the reduction in CPC production.

Oxygen enrichment (sparging bioreactors with oxygen-enriched air) ameliorates oxygen limitation in batch cultures of Aspergillus niger B1-D, and although nutrient utilization rates for both the carbon source and the nitrogen source are higher in oxygen-enriched cultures, excess oxygen does not enhance specific growth rate, instead the 'extra' nutrient consumption is associated with defensive measures. Commencement of oxygen enrichment in early exponential phase leads to a transient rise in proteins showing oxidative damage (carbonylation), and to induction of enhanced proteolytic activity, which points to the antioxidant defense being temporarily overwhelmed, and that A. niger adapts to this oxidative environment by enhancing intracellular proteolytic activity to degrade damaged proteins which might otherwise accumulate under these conditions. Also the energy status of A. niger, reflected by intracellular ATP content, is found to be altered upon the commencement of oxygen enrichment and then reaches much lower levels than in the control. The reasons underlying this are discussed in the context of what is known regarding alternative respiration in industrial fungi.

In the present study, we used a recombinant filamentous fungus strain, Aspergillus niger B1-D, as a model system, and investigated the antioxidant defences in this organism. Our findings indicate that pretreatment with low concentrations of H(2)O(2) completely prevents killing by this oxidant at high concentrations. It shows that A. niger adapts to exposure to H(2)O(2) by reducing growth and inducing a number of antioxidant enzyme activities, including superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, of which the induction of catalase is the most pronounced. Moreover the decline of these antioxidant enzymes activities after H(2)O(2) detoxification, coincides with recommencement of growth. Results from monitoring the extracellular H(2)O(2) concentration clearly indicate a very rapid detoxification rate for H(2)O(2) in adapted A. niger cultures. A mathematical model predicts only very low concentrations of intracellular H(2)O(2) accumulating in such cultures. Our results also show that glutathione plays a role in the oxidative defence against H(2)O(2) in A. niger. On addition of H(2)O(2), the intracellular pool of glutathione increases while the redox state of glutathione becomes more oxidized.

Maca (Lepidium meyenii walp.), a biennial herbaceous plant of the family Brassicae, which is cultivated mainly in the central Andes of Peru, has been used as both a food and a traditional medicine in the region for over 2000 years. The subterranean parts of the plant have long been used as a staple foodstuff by indigenous peoples in the Andean region, but the plant is also valued for its medicinal role. As is usual with many traditional "folk" medicines, many claims have been made regarding the efficacy of maca in treating a wide range of illnesses and medical conditions. However, in the 20th century most scientific attention has been focused in the areas where the pharmacological actions of maca seem most strongly attested, these include, enhancement of sexual drive in humans, increasing overall vigour and energy levels, and increasing sexual fertility in humans and domestic livestock. Since the early days of the 20th century numerous scientific studies have been carried out into the basis of its pharmacological action in these areas. In this review, the composition and pharmacological function of maca are systematically discussed. Additionally, the current discussion surrounding its mode of action in the areas listed above is also presented. (c) 2007 Elsevier Ltd. All rights reserved.

Contamination of food and feed by Aflatoxin B1 (AFB1) is a cause of serious economic and health problems. Different processes have been used to degrade AFB1. In this study, biological degradation of AFB1 was carried out using three Actinomycete species, Rhodococcus erythropolis ATCC 4277, Streptomyces lividans TK 24, and S. aureofaciens ATCC 10762, in liquid cultures. Biodegradation of AFB1 was optimised under a range of temperatures from 25 to 40 °C and pH values of 4.0 to 8.0. An initial concentration of 20 µg/mL of AFB1 was used in this study. The amount of AFB1 remaining was measured against time by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC), coupled with UV and mass spectrometry (LC-MS). All species were able to degrade the AFB1, and no significant difference was found between them. AFB1 remained in the liquid culture for R. erythropolis, S. lividans and S. aureofaciens were 0.81 µg/mL, 2.41 µg/mL and 2.78 µg/mL respectively, at the end of the first 24 h. Degradation occurred at all incubation temperatures and the pH with the optimal conditions for R. erythropolis was achieved at 30 °C and pH 6, whereas for S. lividans and S. aureofaciens the optimum conditions for degradation were 30 °C and pH 5. Analysis of the degradative route indicated that each microorganism has a different way of degrading AFB1. The metabolites produced by R. erythropolis were significantly different from the other two microorganisms. Products of degradation were identified through metabolomic studies by utilizing high-resolution mass spectral data. Mass spectrometric analysis indicated that the degradation of AFB1 was associated with the appearance of a range of lower molecular weight compounds. The pathway of degradation or chemical alteration of AFB1 was followed by means of high resolution Fourier transform mass spectrometry (HR-FTMS) analysis as well as through the MS2 fragmentation to unravel the degradative pathway for AFB1. AFB1 bio-degradation was coupled with the accumulation of intermediates of fatty acid metabolism and glycolysis. A plausible mechanism of degradation of AFB1 by Rhodococcus was hypothesized.

Dissolved carbon dioxide is one variable that changes between scales of bioreactor and may thus have a potential influence on the process as scale alters, and especially so in filamentous fungal cultures. The effects of dCO2 on growth and penicillin production by an industrial strain of Penicillium chrysogenum were examined systematically using a steam sterilisable dCO2 probe and a computerized image analysis system. Although low levels of CO2 stimulated growth (increased biomass, lower mean hyphal growth unit (M.H.G.U.) in lag phase) at the expense of antibiotic synthesis, higher levels led to elevated M.H.G.U.'s in the lag phase and were associated with increased clumping and severely reduced penicillin titre. The adverse effects of dCO2 in these cultures were largely mediated by inhibition of growth and substrate uptake in the lag phase, causing morphological changes which might also contribute to reduced penicillin levels.

The utility of attenuated total reflectance-mid-infrared spectroscopy (ATR-MIR) to monitor the concentrations of the key analytes ammonium, glucose, methyl oleate and biomass in a Streptomyces clavuligerus bioprocess for the synthesis of clavulanic acid was investigated. The cultivation medium was complex and the process fluid (matrix) underwent profound changes as culture growth proceeded, representing a considerable challenge from a spectroscopic viewpoint. Quantitative models were developed using the multivariate statistical technique, partial least square (PLS) for the key analytes over the entire time course of the bioprocess, which were validated externally using samples not incorporated in the original modelling exercise. The reasoning behind the choice of modelling strategy for each analyte is discussed, with particular focus on biomass modelling in order to generate clearer insight into the modelling process in this complex matrix. Despite the heterogeneous nature of the sample matrix, and the complexity of the spectral information arising, at-line models were developed giving low prediction error values for the analytes: ammonium 0.013 g/l; glucose 0.56 g/l; methyl oleate 0.38 g/l and biomass 0.39 g/l, respectively. These findings represent a considerable advance on previous studies using NIR in antibiotic processes, as all key analytes have been successfully modelled here, and the use of ATR-MIR in complex bioprocess fluids has been shown to be a viable method of near real-time monitoring.

The use of at-line NIRS to monitor a high cell density fed-batch baker's yeast bioprocess was investigated. Quantification of the key analytes (biomass, ethanol and glucose) and the product quality indicator (percentage protein content) was studied. Biomass was quantitatively modelled using whole matrix samples (as was percentage protein content). The dominance of the whole matrix spectrum by biomass, and its associated light scattering effects, were overcome by use of filtrate samples and adapted (semi-synthetic) filtrate samples, which allowed successful ethanol and glucose modelling, respectively. Calibrations were rigorously challenged via external validation with large sample sets relative to the calibration sample size, ensuring model robustness and potential practical utility. The standard errors of calibration for biomass, glucose, ethanol and total intracellular protein were (g/l) 1.79, 0.19, 0.79 and 0.91, respectively, comparable to those of the primary assays. The calibration strategies necessary to generate quantitative models for this range of analytes in such a complex high cell density bioprocess fluid are discussed.

The effects of agitation and aeration upon synthesis and molecular weight of the biopolymer gellan were systematically investigated in batch fermenter cultures of the bacterium, Sphingomonas paucimobilis. High aeration rates and vigorous agitation enhanced growth of S. paucimobilis. Although gellan formation occurred mainly in parallel with cell growth, the increase in cells able to synthesise gellan did not always lead to high gellan production. For example, at very high agitation rates (1000 rpm) growth was stimulated at the expense of biopolymer synthesis. Maximal gellan concentration was obtained at 500 rpm agitation and either 1 or 2 vvm aeration (12.3 and 12.4 g/l gellan, respectively). An increase in aeration (from 1 to 2 vvm) enhanced gellan synthesis only at low agitation rates (250 rpm). However, high aeration or dissolved oxygen was not necessary for high gellan synthesis, in fact oxygen limitation always preceded the phase of maximum gellan production and probably enhanced polysaccharide biosynthesis. Some gellan was formed even after glucose exhaustion. This was attributed to the intracellular accumulation of polyhydroxyalkanoates, (such as polyxydroxybutyrate) which were found in S. paucimobilis cells indicating the existence of a carbon storage system, which may contribute to gellan biosynthesis under glucose-limiting conditions. The autolysis of the culture, which occurred at the late stages of the process, seemed to be triggered mainly by limitations in mass (nutrient) transfer, due to the highly viscous process fluid that gradually develops. Rheological measurements generally gave a very good near real time estimate of maximum biopolymer concentration offering the possibility of improved process control relative to time consuming gravimetric assay methods. While mechanical depolymerisation of gellan did not occur, high aeration rates (2 vvm) led to production of gellan of low molecular weight (at either 250 or 500 rpm). This effect of aeration rate upon gellan molecular weight is reported here for the first time, and is important for the properties and applications of gellan. Mechanisms which may have led to this are discussed, but control of molecular weight of the biopolymers is clearly an area needing further research.

Thermophilic aerobic digestion (TAD) is a relatively novel waste processing technique with the potential to improve the safety and nutritional value of many agricultural and food industry wastes for use in animal feed supplementation. TAD of model agricultural waste slurry, at soluble chemical oxygen demand (COD) load of approximately 8.0 g L-1, was implemented to study the effect of the process on crude protein accretion and waste quality. Digestions were carried out under batch conditions at 0.1, 0.25, 0.5 and 1.0 vvm (volume of air per volume of waste slurry per minute) aeration rates and pH 7.0, and at pH 6.0, 7.0, 8.0, 9.0, 9.5 and without pH regulation at 0.5 vvm, all at 55 °C. All reactions were carried out for 156 h using thermophilic populations indigenous to the waste. Digestion at 0.5 and 1.0 vvm led to the greatest degradation of total suspended solids (51.17% and 52.03% respectively) and the highest accretion of crude protein in treated waste (131.28% and 258.94% respectively). Protein content of the digest decreased as digestion pH increased beyond neutral. Slurry soluble protein content decreased with digestion progress, while the ammonium increased early in the process before decreasing progressively. The percentage content of most amino acids in the digest decreased slightly relative to raw waste. The quality of waste protein was comparable with the FAO standard for protein supplement for feed use. Overall, the digested waste was significantly improved relative to the raw waste. With appropriate process optimization TAD has clear potentials for use in the improvement of various wastes for recycling in animal feed.

Gluconobacter suboxydans was used in the biotransformation of d -sorbitol to l -sorbose, a key step in the industrial manufacture of Vitamin C (ascorbic acid). However, despite evidence of the existence of a range of by-products in the bioprocess, relatively little scientific investigation has focused on the factors influencing by-product formation in this complex bioprocess. The aim of the present study was therefore to investigate systematically the extent of by-product formation in this economically important biotransformation process using batch and continuous cultivation methods and up to date analytical techniques and, thus, to arrive at a realistic assessment of the yield of sorbose via this route. The results presented here show that exogenous by-product addition to shake flask and batch cultures detrimentally affects sorbose production and yield, but can have some positive effects in continuous culture. Some inhibition of fructose accumulation by mannitol-1-phosphate addition was noted in shake flask cultures.

Thermophilic aerobic digestion (TAD) of a model agricultural waste, potato peel slurry, at soluble chemical oxygen demand (COD) load equivalent to approximately 8.0 gl(-1), was carried out under batch conditions at 0.5 vvm aeration rate. Digestions were carried out at temperatures of 45, 50, 55, 60 and 65 degrees C (or left unregulated) without pH control to study the effect of digestion temperatures on TAD. The effects of digestion pH on the process were studied at pH 6.0, 7.0, 8.0, 9.0 and 9.5 (and in unregulated control) all at 55 degrees C. Except for digestion at 65 degrees C, which was inoculated extraneously using culture of Bacillus strearothermophilus all reactions were carried out using the populations indigenous to the waste. During digestion at different temperatures, the removal of soluble COD increased with temperature to reach a peak at 60 degrees C before declining slightly, removal of soluble solid (SS) followed similar pattern and reached peak at 65 degrees C being the highest temperature studied, while the degradation of TSS and TS (TSS + TS) decreased with an increase in temperature. Digestion at pH 7.0 was more efficient than at other pH values. Acetate was the predominant volatile fatty acid (VFA) in all the reactions and accounted for up to 90% of the total. Digestion at 60 degrees C led to the greatest accumulation of acetate, and this coincided with the period of highest oxygen uptake, and rapid consumption of soluble carbohydrate. Iso-valerate was also produced at all pH values. Digestion at 55 degrees C and also at pH 7.0 led to rapid and efficient processes with least accumulation of VFA and should be of interest in full-scale processes whenever it is practicable to regulate the digestion pH and temperature. The result of digestion at unregulated pH indicates that gradual adaptation may be used to achieve efficient treatment at elevated pH values. This would be of interest in full-scale processes where it is not practicable to tightly regulate digestion pH, and where the waste is produced at a pH value much higher than neutral.

The use of near infrared spectroscopy (NIRS) was investigated in the context of an efficient high cell density fed-batch industrial Pichia pastoris bioprocess for the production of a therapeutic mammalian protein. This process represented a considerable challenge from the viewpoint of using NIRS to model key analytes because it involved two carbon sources (glycerol and methanol) added at differing rates and times, used a chemically complex medium, and showed a change in liquid phase behaviour due to cell growth. Models for biomass, glycerol, methanol and product were constructed. Different methods of spectral collection and mathematical procedures were used relative to which analyte in the fermentation matrix was being modelled and the rationale behind the model building is clearly described. Regardless of the mode of spectral collection it was essential to consider the changes in modelled analyte concentration relative to changes in other spectral contributors (analytes). The study considerably extends the use of NIRS in fermentation processes to high cell density complex industrial production processes, and comments on how this further developments the technology towards routine in situ NIRS monitoring of bioprocesses.

Thermophilic aerobic digestion (TAD) of a model agricultural waste slurry was carried out in a continuously stirred tank reactor for a total of 156 h at 55 °C. The pH of the digestion was either unregulated or regulated at 6.0, 7.0, 8.0, 9.0 and 9.5 at 0.5 volume of air per volume of waste slurry per minute (vvm) aeration rate. The effect of aeration rate was studied at 0.1, 0.25, 0.5 and 1.0 vvm at pH 7.0. Development of thermophilic populations and amylase and cellulase enzyme activities in the digesting slurry were studied. Thermophiles developed rapidly to reach peak populations in 24 h or earlier. After 96 h, thermophiles were present mostly as spores. They were only slightly affected by aeration rate, but more by digestion pH. Amylase developed rapidly to reach peak activity before declining as rapidly. activity Cellulase was considerably affected by aeration rate but not digestion pH. Amylase profile closely reflected development of microbial activity, and so the progress of TAD. It may be useful as a control tool, either alone or in combination with other process data, for monitoring the TAD of complex waste slurries.

The effects of addition of menadione (MD) to induce elevated intracellular superoxide levels (superoxide stress) and of elevated oxygen availability (oxygen stress) was investigated in batch and chemostat cultures of a recombinant Aspergillus niger (B1-D). Culture responses to the two stressors were clearly distinct in terms of the changes in recombinant protein production, overall protein synthesis, and morphology. MD addition resulted in a significant increase in intracellular O 2 − levels, but gassing with O 2 -enriched air (∼25%) led to decreased intracellular O 2 − levels. Under elevated O 2 availability, the activity of the alternative respiratory pathway was enhanced, reducing endogenous O 2 − generation, but the consequent decreased energy availability in such cultures is proposed to be the main reason leading to the decreased intracellular protein content and foreign protein secretion in batch cultures and chemostat cultures at low dilution rates ( D −1 ). The negative effects of O 2 enrichment on protein synthesis were less pronounced at high D due to the relative dominance of the main respiratory pathway at high growth rates. Under stressed cultivation conditions, the mean number of tips per clump correlated well with the secretion of recombinant protein. Morphological change with O 2 enrichment was not directly caused by the increased O 2 − generation as proposed in some previous studies, but possibly by the significantly altered culture metabolism.

Thermophilic aerobic digestion (TAD) of potato process waste slurry was carried out in a batch process in a continuously stirred tank reactor at 55 °C for a total of 156 h. The pH of the slurry was either unregulated, or regulated at 6.0, 7.0, 8.0, 9.0 and 9.5, and aeration rate was 0.5 vvm. The effect of aeration rate on the digestion process was studied at 0.1, 0.25, 0.5 and 1.0 vvm at pH 7.0 and 55 °C. The development of thermophiles (55 and 65 °C populations) and hydrolytic enzyme activities (protease and xylanase) in the process were monitored. Thermophiles developed rapidly to reach peak populations in 24h or earlier, and remained stable at all fixed pH reactions. The thermophile population was only minimally affected by the aeration rate, and they were present mostly as spores after 96h of digestion. Xylanase enzyme appeared rapidly, reached a peak in approximately 60h, and declined rapidly thereafter. Highest activities were produced in neutral reactions and higher aeration rates. Aeration rates affected protease activity profoundly. The profile of both enzymes closely reflected the development of microbial activity and the overall progress of TAD, in a medium where their substrates were not predominant and not the preferred carbon sources. The relatively simple process of measuring the activities of these enzymes is potentially a more direct measure of the progress of TAD than enumeration of the microbial populations and thus, has greater potential in process monitoring. Copyright © 2003 Society of Chemical Industry

Mid-infrared spectroscopy (MIRS) was used to simultaneously detect and predict concentrations of D-sorbitol and L-sorbose during a Gluconobacter suboxydans biotransformation. Quantitative models for both these compounds were developed for the entire time-course of the process and validated externally using samples not included in the original modelling exercise, giving standard errors of prediction of 3.29 and 3.3% for sorbitol and sorbose, respectively, and a correlation coefficient close to 1.

The application of Fourier Transform near infrared spectroscopy (FT-NIRS) to near real-time monitoring of polysaccharide and biomass concentration was investigated using a gellan-producing strain of Sphingomonas paucimobilis grown in a stirred tank reactor. Successful models for both biomass and gellan were constructed despite the physichochemical complexity of the viscous process fluid. Modelling of biomass proved more challenging than for gellan, partly because of the low range of biomass concentration but a model with a good correlation coefficient (0.94) was formulated based on second derivative spectra. The gellan model was highly satisfactory, with an excellent correlation coefficient (0.98), again based on second derivative spectra. No sample pre-treatment was required and all spectral scanning was carried out on whole broth. Additionally, both models should be robust in practice since both were formulated using low numbers of factors. Thus, the near real time simultaneous monitoring of gellan and biomass in this highly complex matrix using FT-NIRS potentially opens the way to greatly improved process control strategies.

A recombinant strain of Aspergillus niger (B1-D), engineered to produce the marker protein hen egg white lysozyme (HEWL), was investigated with regard to its susceptibility to oxidative stress. The culture response to oxidative stress, produced either by menadione (MD) or by H2O2, was characterised in terms of the intracellular activities of two key defensive enzymes, catalase (CAT) and superoxide dismutase (SOD), in the presence or absence of a range of vitamin supplements. Addition of either ascorbate (Vit C) or α-tocopherol (Vit E) resulted in different responses of A. niger cultures to exogenous H2O2 (1 mM). In the presence of Vit C the activity of SOD was increased by 50% compared to control cultures, whereas incorporation of Vit E appeared to have a “protective” effect against reactive oxygen species (ROS). Continuous addition of menadione (1.2 mmole l−1 h−1) to submerged cultures of A. niger in the bioreactor resulted in an increase of CAT and SOD activities during the exponential phase of growth, whereas enzyme levels remained almost constant during that phase in control cultures. Comparison of these results with the response to oxidative stress caused by addition of exogenous H2O2 and elevated dissolved oxygen tension (DOT) [1] shows that these three stressors are quite distinct.

The robustness of models developed for the near-infrared spectroscopic prediction of mycelial biomass, total sugars, and ammonium in a submerged Penicillium chrysogenum bioprocess was assessed by rigorously challenging them with artificially introduced analyte and background matrix variations, so that analyte concentrations were varied in an invariant matrix and vice versa. The models were also challenged by using a data set from a process operated at a different scale from that used in the original model formulation. Simple univariate and bivariate linear regression models, and partial least-squares (PLS) models with as few factors as three and four, performed sufficiently well for predicting analyte concentrations and were robust with respect to the matrix variations tested. However, models based on relatively weaker absorptions, or those that were likely to be influenced by stronger absorbers present in the same matrix, were vulnerable to changes in the matrix. A change in the scale of operation affected models that would be influenced by biomass, possibly due to an influence of the morphology of the mycelial biomass. An analysis of the loading vectors of some PLS models revealed details that were useful in understanding the type of information modeled and the behavior of these models to the variations tested.

Near-infrared spectroscopy is a promising technique for the rapid monitoring of submerged culture bioprocesses. However, despite the key role of mycelial (filamentous fungal and bacterial) micro-organisms in the manufacture of antibiotics and other valuable therapeutics, there is little information on the application of the technique to monitor mycelial bioprocesses. In part, this is due to the complex and spectroscopically challenging matrices, which result from the growth of these micro-organisms. Moreover, there is a particular lack of any detailed mechanistic information on how models for the prediction of the concentration of key analytes (e.g. biomass, substrates, product) can be constructed, evaluated and improved using the spectral data arising from such complex matrices. We investigated the near-infrared spectra of culture fluid from a submerged fungal bioprocess, for monitoring the concentrations of mycelial biomass and other key analytes. Several empirical models were developed for predicting the concentration of the analytes, using multivariate statistical techniques. Despite the filamentous nature of the biomass and the resulting complexity of the spectral variations, empirical models could be developed for the prediction of this analyte, using biomass ‘specific’ information. SEP values of

Direct detection of intracellular superoxide anion radical (O(2)(.-)) production is of critical importance for investigating the responses of filamentous fungi to oxidative stress in bioprocesses. The purpose of this study is to establish a reliable method to monitor the O(2)(.-) production within pellets of Aspergillus niger. Addition of pure oxygen and the redox cycling agent paraquat to fungal pellet suspensions resulted in a considerable increase in lucigenin-derived chemiluminescence (LDCL). In the presence of exogenous superoxide dismutase (SOD), the LDCL of a disrupted cell solution was inhibited. In contrast, with addition of diethyldithiocarbamate and sodium azide, respectively, the inhibitors of Cu, Zn-SOD and Mn-SOD, an increased LDCL was observed. Further, as a probe, lucigenin can be absorbed and accumulated in fungal pellet within a few minutes. Various pretreatments of the bioreactor sample for the measurement of LDCL, were also investigated in the present study, and the use of intact pellets was adopted here rather than disrupting cells because the latter treatment led to difficulties in LDCL measurement. These results show that lucigenin may be used as a convenient chemiluminescent probe to monitor intracellular production of O(2)(.-) in filamentous fungi, and thus to follow changes in the level of this stressor within fungi

Near infrared spectroscopy (NIRS) was used to monitor an industrial bioprocess for the production of the antibiotic, tylosin, using a segmented modelling approach. Models were built over the entire time course of the fermentation from 0 to 150 h, and also in two distinct phases or segments of the bioprocess from 50 to 100 h (synthetic phase) and from 100 to 150 h (stationary phase). All models were validated externally and the performance of the full range and segmented models compared. The standard error of prediction (SEP) of the segmented models was less in both 50–100 h and 100–150 h and the correlation highest in the 50–100 h range. This would suggest that data segmentation is potentially a useful method of accommodating the impact of the pronounced matrix changes which occur in some bioprocesses in NIRS models for key analytes. While there are many reports on bioprocess monitoring using NIRS, there have been no previous studies on the use of segmented NIR models within a bioprocess as a means of accommodating matrix change.

Organisms of the genus Gluconobacter have been widely utilized within the biotechnology industry for many decades, due to their unique metabolic characteristics. The metabolic features that render Gluconobacter so useful in biotransformation processes, vitamin synthesis, and, as the biological element in sensor systems, are critically evaluated, and the relevance of recent biochemical genetic studies to current and future industrial Gluconobacter processes is discussed. The impact of recombinant gene technology on the status of Gluconobacter processes and the potential use of such techniques in clarifying aspects of the physiology of Gluconobacter is reviewed.

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.

Near-infrared spectroscopy (NIRS) is known to have potential for cost-effective monitoring of bioprocesses. Although this has been demonstrated in many instances and several models have been reported, information regarding the complexity of models required and their utility over extended periods of time is lacking. In the present study, the complexity of the models required for the NIRS prediction of substrate (oil) and product (tylosin) concentration in an industrial bioprocess that employs a physicochemically heterogeneous medium for antibiotic production was assessed. Measurements made by both the diffuse reflectance and transmittance modes were investigated. SEP values for the prediction of the analytes averaged 5% or less, for the successful models, when evaluated using an external validation set, 2 years after the initial model development exercise. Diffuse reflectance measurements showed poorer results, compared to transmittance measurements, especially for monitoring tylosin. In general, this investigation provides evidence to support the fact that models built for the prediction of analytes in a commercial bioprocess that employs a physicochemically complex production medium can be robust in performance over an extended period of time and that simple models based on fewer terms or latent variables can perform well, even in the context of matrices that are relatively complex. It also indicates that sample presentation is likely to be a critical factor in the successful application of NIRS in bioprocess monitoring, which merits further detailed investigation.

The use of near infra-red spectroscopy (NIRS) to monitor a submerged filamentous bacterial bioprocess was investigated. An industrial strain of the filamentous bacterium Streptomyces fradiae was cultured in a 12 litre stirred tank reactor (STR) using a complex medium. This mycelial 4 phase (oil, water, gas and solid) system produced highly complex and variable matrices, therefore monitoring such a complex fluid with NIRS represented a considerable challenge. Nevertheless, successful models for four key analytes (methyl oleate, glucose, glutamate and ammonium) were built at-line (rapid off-line) using NIRS. In the present study, the methods used to formulate, select and validate the models for the key analytes are discussed, with particular emphasis on how the model performance can be critically evaluated. Since previous reports on NIRS in monitoring bioprocesses have either involved simpler matrices, or, in filamentous systems, have not discussed how NIRS models can be critically assessed, the emphasis in the present study on providing an insight into the modelling process in such a complex matrix, may be particularly important to the applicability of NIRS to such industrial bioprocesses.

A recombinant strain of Aspergillus niger (B1-D), engineered to produce the marker protein hen egg white lysozyme, was investigated with regard to its susceptibility to "oxidative stress" in submerged culture in bioreactor systems. The culture response to oxidative stress, produced either by addition of exogenous hydrogen peroxide or by high-dissolved oxygen tensions, was examined in terms of the activities of two key defensive enzymes: catalase (CAT) and superoxide dismutase (SOD). Batch cultures in the bioreactor were generally found to have maximum specific activities of CAT and SOD (Umg x protein(-1)) in the stationary/early-decline phase. Continuous addition of H2O2 (16 mmole L(-1) h(-1)), starting in the early exponential phase, induced CAT but did not increase SOD significantly. Gassing an early exponential-phase culture with O2 enriched (25 vol%) air resulted in increased activities of both SOD and CAT relative to control processes gassed continuously with air, while gassing the culture with 25 vol% O2 enriched air throughout the experiment, although inducing a higher base level of enzyme activities, did not increase the maximum SOD activity obtained relative to control processes gassed continuously with air. The profile of the specific activity of SOD (U mg CDW(-1)) appeared to correlate with dissolved oxygen levels in processes where no H2O2 addition occurred. These findings indicate that it is unsound to use the term "oxidative stress" to encompass a stress response produced by addition of a chemical (H2O2) or by elevated dissolved oxygen levels because the response to each might be quite different.

The use of Fourier transform mid-infrared spectroscopy (FT-MIRS) to predict the concentrations of key analytes in fed-batch cultivations of an industrial strain of Pichia pastoris in a chemical complex medium was investigated. Models for glycerol, methanol (substrates), and product (an heterologous protein) were built, and evaluated. The use of a multi-bounce attenuated total reflectance (HATR) accessory aided spectral acquisition in optically dense samples. Generally, all models were robust and performed well on external validation, using data from processes not present in the original modelling exercise. Substrate models lacked the complexity of some previous IR models, and the models performed adequately even at low analyte concentration (

For decades microbial exopolysaccharides have been invaluable ingredients in the food industry, as well as having many attractive pharmaceutical and chemical applications. Gellan gum is a comparatively new gum elaborated by the Gram-negative bacterium Sphingomonas paucimobilis. Although its physico-chemical properties have been well characterized, the ecology and physiology of Sphingomonas, and the factors influencing the fermentation process for production of this gum have received much less attention. This review focuses on the metabolism and the enzymic activity of this bacterium, as well as the factors that influence gellan production, including process temperature, pH, stirring rate, oxygen transfer, and composition of the production medium. Potential strategies for improving the production process are discussed in the context of processes for the production of other microbial biopolymers, particularly exopolysaccharides. In addition, the importance and potential utility of gellan lyases in modification of gellan and in other applications is critically evaluated.

Thermophilic aerobic digestion(TAD), or liquid composting, is a versatile new process for the treatment and stabilization of high strength wastes of liquid or, perhaps more importantly, slurry consistency. The pattern of inactivation of various pathogenic and indicator organisms was studied using batch digestions under conditions that may be expected to be found in full-scale TAD processes. Rapid inactivation of test populations occurred within the first 10 min from the start of digestion. The inactivation rate was slightly lower when digestions were conducted below 60 degrees C. In some instances, a 'tail' was apparent, possibly indicating the survival of relatively resistant sub-populations particularly in the case of Serratia marcescens and Enterococcus faecalis, or of clumping or attachment of cells to particulate materials. The effect of pH on the inactivation of the test populations depended on the temperature of digestion, but varied with the test population. At 55 degrees C Escherichia coli was more sensitive to temperature effects at pH 7 than at pH 8, but was more sensitive at pH 8, 60 degrees C. The reverse was the case at 60 degrees C for Ent. faecalis. An increase in the solid content of the digesting waste caused a progressive increase in the protection of test organisms from thermal inactivation. Challenging a TAD process with test strains allows (via estimation of D-values) a quantification of the cidal effects of such processes, with a view to manipulating process variables to enhance such effects.

The influence of bioreactor operational conditions on the micromorphology of batch cultures of Aspergillus niger B1-D, containing a hen egg white lysozome (HEWL) marker protein, was examined using computerised image analysis. Significant differences in micromorphology were observed with increased stirrer speed, with shorter organisms with shorter hyphal elements occurring as agitation speed increased, even though mean tip numbers were similar. This may explain the observed increase in the total extracellular protein, since the ratio of synthetic (tip) to non-synthetic zones became increasingly favourable. HEWL concentrations fell above 500rpm, probably due to the effects of DOT (dissolved oxygen tension) on the glucoamylase HEWL fusion. HEWL was susceptible to proteolytic degradation, by native proteases, during the autolytic phase. Such insights may indicate why a gene from one mould expressed in a close relative can give production levels equivalent to levels of native enzymes, while secretion of a gene from a 'distant' source, eg a higher eukaryotic gene, occurs at much lower levels.

Synthesis of the heterologous marker protein hen egg white lysozyme (HEWL) and a native enzyme involved in substrate (starch) degradation (glucoamylase) was studied in chemostat cultures of a recombinant Aspergillus niger (B1-D) at a range of dilution rates. The fungal morphology was quantitatively assessed by means of computerized image analysis (IA) at all dilution rates. Synthesis of the heterologous protein and the native starch processing enzyme was maximal at low dilution rates, and fell as dilution rate ( D ) increased due to a combination of specific induction/repression mechanisms influenced by the residual sugar concentration and the decreased mean residence time of fluid elements in the reactor as D increased. Proteolysis in the culture fluid could not, of itself, explain the decrease in enzyme synthesis as D rose. The fungus synthesized significant amounts of ethanol at all dilution rates, but ethanol synthesis tended to increase with D , due to a steady increase in pellet core diameters and an increasing degree of oxygen limitation. In terms of both the micro- and macromorphology of the culture, increasing the dilution rate tended generally to result in longer “organisms” (i.e., increases in the value of the morphological indices) due to the combined effects of increased extensive growth and decreased hydromechanical damage at higher D. It is clear that the duration of exposure of culture elements to hydromechanical damage may well have had a profound morphological influence. Although there was no correlation between the conventional indices of micromorphology and HEWL/glucoamylase levels, there was a correlation between “percentage active length” of hyphae and total soluble protein concentration, and also between “total concentration of tips” (i.e., tip number per “organism”×biomass concentration) and total soluble protein concentration. This latter quantity (total concentration of tips) may be an adequate, and simpler means of assessing protein-synthesizing capability than more involved double-staining techniques.

This paper reports the effects of Behavioral Family Systems Therapy (BFST) on directly observed interactions between parents and 119 adolescents with diabetes. Families received 3 months' treatment with BFST, an Education and Support Group (ES) or Current Therapy (CT). Multimodal assessments at baseline, posttreatment, and 6- and 12-month follow-ups included tape-recorded family problem-solving discussions scored with the Interaction Behavior Code. Compared with CT and ES, BFST yielded more improvement in family communication and problem-solving skills. Improvements in individual family members' communication were correlated with other improvements in family relationships and adaptation to diabetes, but not with changes in diabetic control. Increased positive and decreased negative reciprocity in family communication and improved family problem solving were associated with improved treatment adherence and adolescent adjustment to IDDM, but not with improved diabetic control.

The process of autolysis in batch cultures of an industrial strain of Penicillium chrysogenum was investigated at a range of stirrer speeds. Autolysis in the cultures was monitored by conventional analysis (biomass decline, NH4+ release), direct measurement of autolysing regions (image analysis), and enzyme activity assays (proteases and β-glucanases). Image analysis measurements provided a sensitive indicator of the onset and progress of autolysis in the culture. Autolysis could be brought about by C or n limitation. Culture history could have a marked effect on the process. Autolysis resulted in degradation of penicillin V and problems with broth filterability. It was closely associated with increased intracellular proteolytic and β-1-3-glucanolytic activities. Although stirrer speed affected both growth and penicillin V production, final mean main hyphal lengths were similar in autolysing cultures. This indicated a possible role of an intrinsic characteristic in fragmentation.

The process of cellular autolysis was studied in an industrial strain of Penicillium chrysogenum by a range of methods, including assessment of biomass decline, NH+4 release, changes in culture apparent viscosity, and by means of a quantitative assessment of changes in micromorphology using a computerized image analysis system. The pattern of total intracellular proteolytic and β-1,3-glucanolytic activity in the culture was also examined. The overall aim was to identify a suitable method, or methods, for examining the extent of autolysis in fungal cultures. Autolysis was studied in submerged batch processes, where DOT was maintained above 40% saturation (non-O2-limited), and, under O2-limited conditions. Both N and O2 limitation promoted extensive culture autolysis. Image analysis techniques were perhaps the most sensitive method of assessing the progress of autolysis in the culture. Autolytic regions within some hyphae were apparent even during growth phase, but became much more widespread as the process proceeded. The early stages of autolysis involved continued energy source consumption, increased carbon dioxide evolution rate, degradation of penicillin, and decreased broth filterability. Later stages involved widespread mycelial fragmentation, with some regrowth (cryptic growth) occurring in non-O2-limited cultures. Intracellular proteolytic activity showed two peaks, one during the growth phase, and the other during autolysis. Autolysis was also associated with a distinct peak in β-1,3-glucanolytic activity, indicating that degradation of cell wall matrix polymers may be occurring during autolysis in this strain of P. chrysogenum. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 297–305, 1998.

Two physically distinct exopolysaccharide fractions were extracted from cultures of the polymorphic fungus Aureobasidium pullulans grown in a stirred tank bioreactor and compared with a commercial sample of pullulan. The two fractions were characterised using elemental analysis, infrared spectroscopy, GC-MS and gel permeation chromatography. Both fractions were found to be structurally and compositionally identical, predominantly linear glucans containing α-1,4- and α-1,6-linkages. From these results, it is clear that the differing physical behaviour and appearance of the two fractions can be attributed solely to differences in molecular mass. Possible mechanisms by which the two fractions may have occurred are discussed in the context of other reports concerning exopolysaccharide synthesis by this fungus.

This review focuses on recent progress in the technology of high throughput (HTP) cultivation and its increasing application in quality by design (QbD) -driven bioprocess development. Several practical HTP strategies aimed at shortening process development (PD) timelines from DNA to large scale processes involving commercially available HTP technology platforms, including microtiter plate (MTP) culture, micro-scale bioreactors, and in parallel fermentation systems, etc., are critically reviewed in detail. This discussion focuses upon the relative strengths and weaknesses or limitations of each of these platforms in this context. Emerging prototypes of micro-bioreactors reported recently, such as milliliter (mL) scale stirred tank bioreactors, and microfludics integrated micro-scale bioreactors, and their potential for practical application in QbD-driven HTP process development are also critically appraised. The overall aim of such technology is to rapidly gain process insights, and since the analytical technology deployed in HTP systems is critically important to the achievement of this aim, this rapidly developing area is discussed. Finally, general future trends are critically reviewed.

The formation of ethanol by the polymorphic fungus Aureobasidium pullulans was examined under a range of culture pH values and aeration conditions. Although culture pH had a profound effect on fungal morphology, with a greatly increased proportion of the biomass in the unicellular (yeast-like) form at pH 6.5, there appeared to be no direct link between morphological form and ethanol formation. The levels of ethanol noted may have influenced the morphology. Cessation of aeration rapidly led to a halt in growth and exopolysaccharide synthesis, while ethanol synthesis proceeded rapidly. The ethanol formed could be re-oxidised under conditions where the available carbon source was depleted. Two distinct exopolysaccharides of different molecular weights were recovered from the culture fluids. Overall, the molecular weight of both declined with process time.