Fermentation and microbial physiology

Scientific publications - Fermentation and microbial physiology

Albaek, M.O., Gernaey, K. V., Hansen, M. S., Stocks, S. M
Modeling Enzyme Production With Aspergillus oryzae in Pilot Scale Vessels With Different Agitation, Aeration, and Agitator Types”
Biotechnology and Bioengineering 108(8) pp 1828- 1840 (2011)


The purpose of this article is to demonstrate how a model can be constructed such that the progress of a submerged fed-batch fermentation of a filamentous fungus can be predicted with acceptable accuracy. The studied process was enzyme production with Aspergillus oryzae in 550 L pilot plant stirred tank reactors. Different conditions of agitation and aeration were employed as well as two different impeller geometries. The limiting factor for the productivity was oxygen supply to the fermentation broth, and the carbon substrate feed flow rate was controlled by the dissolved oxygen tension. In order to predict the available oxygen transfer in the system, the stoichiometry of the reaction equation including maintenance substrate consumption was first determined. Mainly based on the biomass concentration a viscosity prediction model was constructed, because rising viscosity of the fermentation broth due to hyphal growth of the fungus leads to significant lower mass transfer towards the end of the fermentation process. Each compartment of the model was shown to predict the experimental results well. The overall model can be used to predict key process parameters at varying fermentation conditions.

Albaek, M.O., Gernaey, K. V., Stocks, S.
"Gassed and ungassed power draw in a pilot scale 550 litre fermentor retrofitted with up-pumping hydrofoil B2 impellers in media of different viscosity and with very high power draw”
Chemical Engineering Science 63 (24) pp. 5813 – 5820 (2008)


A number of modern impellers have been designed in the pursuit of an alternative to the traditional Rushton turbine, which has a number of well recognized deficiencies. A dual up-pumping combination of the Hayward Tyler B2 (former APV-B2 or simply B2), a high solidity ratio hydrofoil impeller, was retrofitted using traditional methods to a pilot scale fermentor based on cited reference studies of the impeller performance. Using water as a media and comparatively low power draws, the B2 impeller has previously been shown to have good gas handling properties and a low ungassed power number allowing for use at high impeller-to-tank diameter ratio. In the present study a power characterization of the B2 impeller was undertaken in order to extend the available data to viscous media resembling fermentation broths and to very high power draws. Power characterizations were carried out with specific energy input rates up to 12.9 kW/m3 using different shear-thinning media.
The ungassed power number of the B2 impeller was determined (3.3 for power draws in the range 0–11.6 kW/m3) and our findings confirm those of previous studies and extend the trends to media of high viscosity. Upon aeration the B2 impellers loose little power compared to the traditional impeller even when applying a very high power draw (10–20% power loss at 450 rpm and 1.28 vvm corresponding to 11.6 kW/m3). Torque fluctuations are found to be small (<5%) for this impeller at high power draw and high viscosity media. Finally it is shown that the B2 impeller can be retrofitted to pilot scale fermentors using traditional methods with a high degree of confidence.


 Petersen, N., Stocks, S., Gernaey, K.V.

"Multivariate models for prediction of rheological characteristics of filamentous fermentation broth from the size distribution"

Biotechnology and Bioengineering, 100 (1), pp. 61-71. (2008)

The main purpose of this article is to demonstrate that principal component analysis (PCA) and partial least squares regression (PLSR) can be used to extract information from particle size distribution data and predict rheological properties. Samples from commercially relevant Aspergillus oryzae fermentations conducted in 550 L pilot scale tanks were characterized with respect to particle size distribution, biomass concentration, and rheological properties. The rheological properties were described using the Herschel-Bulkley model. Estimation of all three parameters in the Herschel-Bulkley model (yield stress (τy), consistency index (K), and flow behavior index (n)) resulted in a large standard deviation of the parameter estimates. The flow behavior index was not found to be correlated with any of the other measured variables and previous studies have suggested a constant value of the flow behavior index in filamentous fermentations. It was therefore chosen to fix this parameter to the average value thereby decreasing the standard deviation of the estimates of the remaining rheological parameters significantly. Using a PLSR model, a reasonable prediction of apparent viscosity (μapp), yield stress (τy), and consistency index (K), could be made from the size distributions, biomass concentration, and process information. This provides a predictive method with a high predictive power for the rheology of fermentation broth, and with the advantages over previous models that τy and K can be predicted as well as μapp. Validation on an independent test set yielded a root mean square error of 1.21 Pa for τy, 0.209 Pa sn for K, and 0.0288 Pa s for μapp, corresponding to R2 = 0.95, R2 = 0.94, and R2 = 0.95 respectively. © 2007 Wiley Periodicals, Inc.

Bodizs, L., Titica, M., Faria, N., Srinivasan, B., Dochain, D., Bonvin, D.

"Oxygen control for an industrial pilot-scale fed-batch filamentous fungal fermentation"

Journal of Process Control, 17 (7), pp. 595-606. (2007)

Industrial filamentous fungal fermentations are typically operated in fed-batch mode. Oxygen control represents an important operational challenge due to the varying biomass concentration. In this study, oxygen control is implemented by manipulating the substrate feed rate, i.e. the rate of oxygen consumption. It turns out that the setpoint for dissolved oxygen represents a trade-off since a low dissolved oxygen value favors productivity but can also induce oxygen limitation. This paper addresses the regulation of dissolved oxygen using a cascade control scheme that incorporates auxiliary measurements to improve the control performance. The computation of an appropriate setpoint profile for dissolved oxygen is solved via process optimization. For that purpose, an existing morphologically structured model is extended to include the effects of both low levels of oxygen on growth and medium rheological properties on oxygen transfer. Experimental results obtained at the industrial pilot-scale level confirm the efficiency of the proposed control strategy but also illustrate the shortcomings of the process model at hand for optimizing the dissolved oxygen setpoints. © 2007 Elsevier Ltd. All rights reserved.

Hewitt, C.J., Papapanagiotou, P.A., Quinn, H., Molitor, J.P., Stocks, S.M., Nienow, A.W.

"The use of emulsification technologies to enhance rapeseed oil consumption during industrial Streptomyces rimosus fed-batch fermentations"

Chemical Engineering Research and Design, 85 (7 A), pp. 1072-1078. (2007)

Phase inversion temperature (PIT) and self-emulsification (SE) technologies were used to produce novel forms of oil feed for fed-batch fermentations. These novel forms were supplied with the aim of improving both oil utilisation and product titre whilst reducing fermentation broth viscosity. The study involved industrial Streptomyces rimosus fermentations fed with rape-seed oil (RSO) for the production of oxytetracycline. In all cases, oil utilisation increased when compared to the control process. Additionally, a very significant decrease (∼50%) in the final concentration of residual oil was also achieved when either the PIT or self-emulsifying technologies were used. Further, a significant decrease (∼75%) in the culture broth viscosity was also measured when using the self-emulsifying method. It was concluded that the measurable increase in the utilization of oil was due to the formation of smaller and more stable oil droplets when using such technologies and/or the lower broth viscosity, which increased the mass transfer from the complex fermentation medium to the cells. In all cases, the final oxytetracycline concentration and time of attenuation was not significantly different for any of the fermentation processes. © 2007 Institution of Chemical Engineers.

Jørgensen, T.R., vanKuyk, P.A., Poulsen, B.R., Ruijter, G.J.G., Visser, J., Iversen, J.J.L.

"Glucose uptake and growth of glucose-limited chemostat cultures of Aspergillus niger and a disruptant lacking MstA, a high-affinity glucose transporter"

Microbiology, 153 (6), pp. 1963-1973. (2007)

This is a study of high-affinity glucose uptake in Aspergillus niger and the effect of disruption of a high-affinity monosaccharide-transporter gene, mstA. The substrate saturation constant (Ks) of a reference strain was about 15 μM in glucose-limited chemostat culture. Disruption of mstA resulted in a two- to fivefold reduction in affinity for glucose and led to expression of a low-affinity glucose transport gene, mstC, at high dilution rate. The effect of mstA disruption was more subtle at low and intermediate dilution rates, pointing to some degree of functional redundancy in the high-affinity uptake system of A. niger. The mstA disruptant and a reference strain were cultivated in glucose-limited chemostat cultures at low, intermediate and high dilution rate (D=0.07 h-1, 0.14 h-1 and 0.20 h-1). Mycelium harvested from steady-state cultures was subjected to glucose uptake assays, and analysed for expression of mstA and two other transporter genes, mstC and mstF. The capacity for glucose uptake (vmax) of both strains was significantly reduced at low dilution rate. The glucose uptake assays revealed complex uptake kinetics. This impeded accurate determination of maximum specific uptake rates (vmax) and apparent affinity constants (Kmapp) at intermediate and high dilution rate. Two high-affinity glucose transporter genes, mstA and mstF, were expressed at all three dilution rates in chemostat cultures, in contrast to batch culture, where only mstC was expressed. Expression patterns of the three transporter genes suggested differential regulation and functionality of their products. © 2007 SGM.

Eriksen, N.T., Riisgård, F.K., Gunther, W.S., Lønsmann Iversen, J.J.

"On-line estimation of O2 production, CO2 uptake, and growth kinetics of microalgal cultures in a gas-tight photobioreactor"

Journal of Applied Phycology, 19 (2), pp. 161-174. (2007)

Growth of the green algae Chlamydomonas reinhardtii and Chlorella sp. in batch cultures was investigated in a novel gas-tight photobioreactor, in which CO2, H2, and N2 were titrated into the gas phase to control medium pH, dissolved oxygen partial pressure, and headspace pressure, respectively. The exit gas from the reactor was circulated through a loop of tubing and re-introduced into the culture. CO2 uptake was estimated from the addition of CO2 as acidic titrant and O 2 evolution was estimated from titration by H2, which was used to reduce O2 over a Pd catalyst. The photosynthetic quotient, PQ, was estimated as the ratio between O2 evolution and CO 2 up-take rates. NH4+, NO2-, or NO3- was the final cell density limiting nutrient. Cultures of both algae were, in general, characterised by a nitrogen sufficient growth phase followed by a nitrogen depleted phase in which starch was the major product. The estimated PQ values were dependent on the level of oxidation of the nitrogen source. The PQ was 1 with NH4+ as the nitrogen source and 1.3 when NO3- was the nitrogen source. In cultures grown on all nitrogen sources, the PQ value approached 1 when the nitrogen source was depleted and starch synthesis became dominant, to further increase towards 1.3 over a period of 3-4 days. This latter increase in PQ, which was indicative of production of reduced compounds like lipids, correlated with a simultaneous increase in the degree of reduction of the biomass. When using the titrations of CO2 and H2 into the reactor headspace to estimate the up-take of CO2, the production of O2, and the PQ, the rate of biomass production could be followed, the stoichiometrical composition of the produced algal biomass could be estimated, and different growth phases could be identified. © 2006 Springer Science+Business Media, Inc.

Mead, D., Pearson, D., Devine, M.

"Recombinant human albumin: Applications as a biopharmaceutical excipient"

Innovations in Pharmaceutical Technology, (22), pp. 42-44. (2007)


Wigley, A., Wilkinson, D., Mead, D.

"Managing scale-up of recombinant proteins"

Genetic Engineering and Biotechnology News, 27 (2), pp. 40+42. (2007)


Mortensen, P.P., Bro, R.

"Real-time monitoring and chemical profiling of a cultivation process"

Chemometrics and Intelligent Laboratory Systems, 84 (1-2 SPEC. ISS.), pp. 106-113. (2006)

A method for at-line quality assessment of a cultivation process is developed in order to (1) enable improved process control, (2) enable faster detection of batch end point, and (3) enable immediate quality assessment of final product. Fluorescence excitation-emission measurements are used because they are known to reflect important properties of the fermentation process. Focus is also on important sampling issues-mainly structurally sub-optimal primary sampling methods affecting the representativity obtainable relative to the lot characteristics. Several different calibration approaches are investigated for predicting product quality (enzymatic activity). The fluorescence data are also modeled by a PARAFAC model, providing a chemically interpretable visualization of the process variation thereby enhancing the possibilities for gaining in-depth process understanding. The results of our investigations are that enzyme activity can be predicted directly from fluorescence measurements with an uncertainty comparable to the uncertainty of the chemical reference analysis for the enzyme activity. Chemical profiling of the cultivation process using PARAFAC verifies basic understanding of the cultivation process. An enzyme marker profile as well as a tryptophan (protein marker) profile is identified. © 2006 Elsevier B.V. All rights reserved.

Agbogbo, F.K., Coward-Kelly, G., Torry-Smith, M., Wenger, K.S.

"Fermentation of glucose/xylose mixtures using Pichia stipitis"

Process Biochemistry, 41 (11), pp. 2333-2336. (2006)

Different synthetic glucose/xylose mixtures (60 g/L total sugar) were fermented into ethanol using P. stipitis CBS 6054. Glucose was the preferred substrate in the glucose/xylose mixtures. The high glucose fractions had higher cell biomass production rate and therefore higher substrate consumption rate and ethanol production rate compared to high xylose fractions. However, the high xylose fractions had a slightly higher ethanol yields compared to the high glucose fractions because the substrate was channeled into ethanol production rather the cell biomass. The maximum ethanol concentration was 22.7 and 24.3 g/L in 60 g/L glucose and xylose media, respectively. Using the same initial cell concentration of 2 g/L, fermentation was complete after 96 h of fermentation in 100% glucose (60 g/L), and after 120 h of fermentation in 100% xylose (60 g/L). © 2006 Elsevier Ltd. All rights reserved.

Diano, A., Bekker-Jensen, S., Dynesen, J., Nielsen, J.

"Polyol synthesis in Aspergillus niger: Influence of oxygen availability, carbon and nitrogen sources on the metabolism"

Biotechnology and Bioengineering, 94 (5), pp. 899-908. (2006)

Polyol production has been studied in Aspergillus niger under different conditions. Fermentations have been run using high concentration of glucose or xylose as carbon source and ammonium or nitrate as nitrogen source. The growth of biomass, as freely dispersed hyphae, led to an increase of medium viscosity and hereby a decrease in mass transfer, especially oxygen transfer. The consequence was a decrease in DOT and the occurrence of a switch between fully aerobic conditions and oxygen-limited conditions. Metabolite quantification showed that polyols were the main metabolic products formed and represented up to 22% of the carbon consumed in oxygen-limited conditions. The polyol concentration and the polyol pattern depended strongly on the environmental conditions. This is due to a complex regulation of polyol production and to the fact that each polyol can fulfill different functions. In this study, erythritol, xylitol, and arabitol were produced as carbon storage compounds when the flux through the PP pathway exceeded the need in ribulose-5-phosphate for the biomass synthesis. Glycerol, erythritol, and xylitol seem to be involved in osmoregulation. Mannitol was produced when the catabolic reduction of charge was high. Its production involves the enzyme NAD-dependent mannitol-1-phosphate dehydrogenase and seems to be the main cytosolic route for the NADH reoxidation during oxygen limitation. © 2006 Wiley Periodicals, Inc.

Haack, M.B., Olsson, L., Hansen, K., Lantz, A.E.

"Change in hyphal morphology of Aspergillus oryzae during fed-batch cultivation"

Applied Microbiology and Biotechnology, 70 (4), pp. 482-487. (2006)

Industrial enzymes are often produced by filamentous fungi in fed-batch cultivations. During cultivation, the different morphological forms displayed by the fungi have an impact on the overall production. The morphology of a recombinant lipase producing Aspergillus oryzae strain was investigated during fed-batch cultivations. During the exponential batch phase of the fed-batch cultivations, the average hyphal length increased as did the number of tips per hyphal element. Most striking was the finding that the diameter of the hyphal elements increased with an average factor of 1.5 during the batch phase from 2.8-2.9 up to 4.0-4.4 μm. The diameter of the hyphal elements remained constant, around 4 μm, after the feed was started. However, the diameter of the immediate hyphal tip, where the enzyme secretion is thought to take place, increased dramatically with up to a factor 2.5 during the feeding period. The expression of the recombinant lipase was induced by the feeding with maltose, and an increase in lipase activity was seen in parallel to a swelling of the tips. The results indicate that the two events are linked as a return to normal growth was observed upon cessation of production due to oxygen limitations. © Springer-Verlag 2005.

Sianidis, G., Pozidis, C., Becker, F., Vrancken, K., Sjoeholm, C., Karamanou, S., Takamiya-Wik, M., Van Mellaert, L., Schaefer, T., Anné, J., Economou, A.

"Functional large-scale production of a novel Jonesia sp. xyloglucanase by heterologous secretion from Streptomyces lividans"

Journal of Biotechnology, 121 (4), pp. 498-507. (2006)

The gene encoding a novel xyloglucanase (Xeg) belonging to family 74 glycoside hydrolases was isolated from a Jonesia sp. strain through functional screening in Escherichia coli. The encoded xyloglucanase is a protein of 972 aminoacyl residues with a 23 residue aminoterminal signal peptide. Over-expression of Xeg in B. subtilis or E. coli failed. In contrast, Xeg was successfully over-expressed and secreted in Streptomyces lividans TK24. To this end Xeg was fused C-terminally to the secretory signal peptide of the subtilisin inhibitor protein (vsi) from Streptomyces venezuelae. The native Xeg signal peptide derived from Jonesia sp. is only poorly functional in S. lividans. Under optimal growth conditions, significant amounts of mature Xeg (100-150 mg/l) are secreted in the spent growth media. A protocol to rapidly purify Xeg to homogeneity from culture supernatants was developed. Biophysical and biochemical analyses indicate that the enzyme is intact, stable and fully functional. Xeg is the longest heterologous polypeptide shown to be secreted from S. lividans. This study further validates use of S. lividans for production of active heterologous proteins and demonstrates that heterologous polypeptides of up to 100 kDa are also tractable by this system.

Johansson, L., Lindskog, A., Silfversparre, G., Cimander, C., Nielsen, K.F., Lidén, G.

"Shikimic acid production by a modified strain of E. coli (W3110.shik1) under phosphate-limited and carbon-limited conditions"

Biotechnology and Bioengineering, 92 (5), pp. 541-552. (2005)

Shikimic acid is one of several industrially interesting chiral starting materials formed in the aromatic amino acid pathway of plants and microorganisms. In this study, the physiology of a shikimic acid producing strain of Escherichia coli (derived from W3110) deleted in aroL (shikimic acid kinase II gene), was compared to that of a corresponding control strain (W3110) under carbon-and phosphate-limited conditions. For the shikimic acid producing strain (referred to as W3110.shik1), phosphate limitation resulted in a higher yield of shikimic acid (0.059 ± 0.012 vs. 0.024 ± 0.005 c-mol/c-mol) and a lower yield of by-products from the shikimate pathway, when compared to carbon-limited condition. The yield of the by-product 3-dehydroshikimic acid (DHS) decreased from 0.076 ± 0.028 to 0.022 ± 0.001 c-mol/c-mol. Several other by-products were only detected under carbon-limited conditions. The latter group included 3-dehydroquinic acid (0.021 ± 0.021 c-mol/c-mol), quinic acid (0.012 ± 0.005 c-mol/c-mol), and gallic acid (0.002 ± 0.001 c-mol/c-mol). For both strains, more acetate was produced under phosphate than the carbon-limited case. Considerable cell lysis was found for both strains but was higher for W3110.shik1, and increased for both strains under phosphate limitation. The advantages of the latter condition in terms of an increased shikimic acid yield was thus counteracted by an increased cell lysis, which may make downstream processing more difficult. © 2005 Wiley Periodicals, Inc. 

Jürgen, B., Tobisch, S., Wümpelmann, M., Gördes, D., Koch, A., Thurow, K., Albrecht, D., Hecker, M., Schweder, T.

"Global expression profiling of Bacillus subtilis cells during industrial-close fed-batch fermentations with different nitrogen sources"

Biotechnology and Bioengineering, 92 (3), pp. 277-298. (2005)

A detailed gene expression analysis of industrial-close Bacillus subtilis fed-batch fermentation processes with casamino acids as the only nitrogen source and with a reduced casamino acid concentration but supplemented by ammonia was carried out. Although glutamine and arginine are supposed to be the preferred nitrogen sources of B. subtilis, we demonstrate that a combined feeding of ammonia and casamino acids supports cell growth under fed-batch fermentation conditions. The transcriptome and proteome analyses revealed that the additional feeding of ammonia in combination with a reduced amino acid concentration results in a significantly lower expression level of the glnAR or tnrA genes, coding for proteins, which are mainly involved in the nitrogen metabolism of B. subtilis. However, the mRNA levels of the genes of the ilvBHC-leuABD and hom-thrCB operons were significantly increased, indicating a valine, leucine, isoleucine, and threonine limitation under these fermentation conditions. In contrast, during the fermentation with casamino acids as the only nitrogen source, several genes, which play a crucial role in nitrogen metabolism of B. subtilis(e.g., glnAR, nasCDE, nrgAB.and ureAB), were up-regulated, indicating a nitrogen limitation under these conditions. Furthermore, increased expression of genes, which are involved in motility and chemotaxis (e.g., hag, fliT) and in acetoin metabolism (e.g., acoABCL), was determined during the fermentation with the mixed nitrogen source of casamino acids and ammonia, indicating a carbon limitation under these fermentation conditions. Under high cell density and slow growth rate conditions a weak up-regulation of autolysis genes could be observed as well as the induction of a number of genes involved in motility, chemotaxis and general stress response. Results of this study allowed the selection of marker genes, which could be used for the monitoring of B. subtilis fermentation processes. The data suggest for example acoA as a marker gene for glucose limitation or glnA as an indicator for nitrogen limitation. © 2005 Wiley Periodicals, Inc.

Aiello-Mazzarri, C., Coward-Kelly, G., Agbogbo, F.K., Holtzapple, M.T.

"Conversion of municipal solid waste into carboxylic acids by anaerobic countercurrent fermentation: Effect of using intermediate lime treatment"

Applied Biochemistry and Biotechnology, 127 (2), pp. 79-93. (2005)

Municipal solid waste (MSW) and sewage sludge (SS) were combined and anaerobically converted into carboxylate salts by using a mixed culture of acid-forming microorganisms. MSW is an energy source and SS is a source of nutrients. In this study, MSW and SS were combined, so they complemented each other. Four fermentors were arranged in series for a countercurrent fermentation process. In this process, the solids and liquid were transferred in opposite directions, with the addition of fresh biomass to fermentor 1 and fresh liquid media to fermentor 4. An intermediate lime treatment of solids exiting fermentor 3 before entering fermentor 4 was applied to improve the product acid concentration from the untreated MSW/SS fermentations. All fermentations were performed under anaerobic conditions at 40°C. Calcium carbonate was added to neutralize the carboxylic acids and to control the pH. Iodoform was used as a methanogen inhibitor. Carboxylic acid concentration and gas composition were determined by gas chromatography. Substrate conversion was measured by volatile solids loss, and carboxylic acid productivity was calculated as the function of the total carboxylic acids produced, the amount of liquid in all fermentors, and time. The addition of intermediate lime treatment increased product concentration and conversion by approx 30 and 15%, respectively. The highest carboxylic acid concentrations for untreated MSW/SS fermentations with and without intermediate lime treatment were 22.2 and 17.7 g of carboxylic acid/L of liquid, respectively. These results confirm that adding a treatment step between fermentor 3 and fermentor 4 will increase the digestibility and acid productivity of the fermentation. Copyright © 2005 by Humana Press Inc. All rights of any nature whatsoever reserved.

Kvist, T., Thyregod, P.

"Using evolutionary operation to improve yield in biotechnological processes"

Quality and Reliability Engineering International, 21 (5), pp. 457-463. (2005)

In the biotechnological industry, production is often characterized by relatively few larger batches. In the design stages of a new process, the use of statistical methods for experimentation can provide invaluable information about the process. However, it is frequently found that optimum conditions in the laboratory or pilot plant give lower yields when transferred to full-scale production. This fact is due to scale-up effects and to the large inherent variations when dealing with biological material and processes such as fermentation. In full-scale production there is not the same freedom to experimentation as at the laboratory scale. Simple one factor at a time trials are predominant when attempting to improve the yield at the production scale. In this way it is possible to control the outcome such that it still meets the requirements. However, this is a very inefficient way to perform experiments. The requirements for an alternative experimental procedure is that it should be robust to non-controllable variations, it should contain automatic safeguards to ensure that unsatisfactory material is not manufactured and it should be possible to make decisions during the trial. Furthermore, it is important that the planning stage is short and the interpretation straightforward. The method of evolutionary operation (EVOP) suggested by George Box fulfilled these requirements. We present the implementation of EVOP in a major Danish biotechnological company. An example is presented where the method was used in the fermentation process of an industrial enzyme. In the particular example, process yield was improved by 45%. Copyright © 2005 John Wiley & Sons, Ltd.

De Maré, L., Velut, S., Ledung, E., Cimander, C., Norrman, B., Karlsson, E.N., Holst, O., Hagander, P.

"A cultivation technique for E. coli fed-batch cultivations operating close to the maximum oxygen transfer capacity of the reactor"

Biotechnology Letters, 27 (14), pp. 983-990. (2005)

A cultivation strategy combining the advantages of temperature-limited fed-batch and probing feeding control is presented. The technique was evaluated in fed-batch cultivations with E. coli BL21(DE3) producing xylanase in a 3 liter bioreactor. A 20% increase in cell mass was achieved and the usual decrease in specific enzyme activity normally observed during the late production phase was diminished with the new technique. The method was further tested by growing E. coli W3110 in a larger bioreactor (50 l). It is a suitable cultivation technique when the O2 transfer capacity of the reactor is reached and it is desired to continue to produce the recombinant protein. © Springer 2005.

Schmidt, R.A., Wiebe, M.G., Eriksen, N.T.

"Heterotrophic high cell-density fed-batch cultures of the phycocyanin-producing red alga Galdieria sulphuraria"

Biotechnology and Bioengineering, 90 (1), pp. 77-84. (2005)

Growth and phycocyanin production in batch and fed-batch cultures of the microalga Galdieria sulphuraria 074G, which was grown heterotrophically in darkness on glucose, fructose, sucrose, and sugar beet molasses, was investigated. In batch cultures, specific growth rates and yields of biomass dry weight on the pure sugars were 1.08-1.15 day-1 and 0.48-0.50 g g-1, respectively. They were slightly higher when molasses was the carbon source. Cellular phycocyanin contents during the exponential growth phase were 3-4 mg g-1 in dry weight. G. sulphuraria was able to tolerate concentrations of glucose and fructose of up to 166 g L-1 (0.9 M) and an ammonium sulfate concentration of 22 g L-1 (0.17 M) without negative effects on the specific growth rate. When the total concentration of dissolved substances in the growth medium exceeded 1-2 M, growth was completely inhibited. In carbon-limited fed-batch cultures, biomass dry weight concentrations of 80-120 g L-1 were obtained while phycocyanin accumulated to concentrations between 250 and 400 mg L-1. These results demonstrate that G. sulphuraria is well suited for growth in heterotrophic cultures at very high cell densities, and that such cultures produce significant amounts of phycocyanin. Furthermore, the productivity of phycocyanin in the heterotrophic fed-batch cultures of G. sulphuraria was higher than is attained in outdoor cultures of Spirulina platensis, where phycocyanin is presently obtained. © 2005 Wiley Periodicals, Inc.

Stocks, S.M., Cooke, M., Heggs, P.J.

"Inverted hollow spinning cone as a device for controlling foam and hold-up in pilot scale gassed agitated fermentation vessels"

Chemical Engineering Science, 60 (8-9 SPEC. ISS.), pp. 2231-2238. (2005)

The effectiveness of an inverted hollow spinning cone (IHSC) for controlling foam and liquid hold-up is demonstrated in pilot scale equipment for a currently commercially exploited recombinant Bacillus fermentation with the characteristically challenging foaming and hold-up issues of this genus. The cone rotates on the existing agitator shaft at the normal production operating level. A series of 9 trials were conducted in 550-1 pilot scale fermenters: 2 fermentations without foam control agent (FCA) addition, 4 controls with FCA addition on demand, and three with the cone as the sole method of level control. Batches running without FCA addition or the use of an IHSC lost 52 ± 15kg of broth through the exhaust system. Batches with FCA addition on demand or the IHSC ended with a statistically indistinguishable filling of 308 ± 6kg and 319 ± 13kg, respectively. Process performance in terms of enzyme titre was not affected. Such encouraging results indicate great potential savings in both fermentation and recovery costs since FCA addition can be completely avoided. A full-scale production evaluation is justified. © 2004 Elsevier Ltd. All rights reserved.

Bhargava, S., Wenger, K.S., Rane, K., Rising, V., Marten, M.R.

Effect of cycle time on fungal morphology, broth rheology, and recombinant enzyme productivity during pulsed addition of limiting carbon source

Biotechnology and Bioengineering, 89 (5), pp. 524-529. (2005)

For many years, high broth viscosity has remained a key challenge in large-scale filamentous fungal fermentations. In previous studies, we showed that broth viscosity could be reduced by pulsed addition of limiting carbon during fed-batch fermentation. The objective in this study was to determine how changing the frequency of pulsed substrate addition affects fungal morphology, broth rheology, and recombinant enzyme productivity. To accomplish this, a series of duplicate fed-batch fermentations were performed in 20-L fermentors with a recombinant glucoamylase producing strain of Aspergillus oryzae. The total cycle time for substrate pulsing was varied over a wide range (30-2, 700 s), with substrate added only during the first 30% of each cycle. As a control, a fermentation was conducted with continuous substrate feeding, and in all fermentations the same total amount of substrate was added. Results show that the total biomass concentration remained relatively unaltered, while a substantial decrease in the mean projected area of fungal elements (i.e., average size) was observed with increasing cycle time. This led to reduced broth viscosity and increased oxygen uptake rate. However, high values of cycle time (i.e., 900-2, 700 s) showed a significant increase in fungal conidia formation and significantly reduced recombinant enzyme productivity, suggesting that the fungi channeled substrate to storage compounds rather than to recombinant protein. In addition to explaining the effect of cycle time on fermentation performance, these results may aid in explaining the discrepancies observed on scale-up to larger fermentors. © 2005 Wiley Periodicals, Inc.

Hornbæk, T., Jakobsen, M., Dynesen, J., Nielsen, A.K.

"Global transcription profiles and intracellular pH regulation measured in Bacillus licheniformis upon external pH upshifts"

Archives of Microbiology, 182 (6), pp. 467-474. (2004)

For optimization of propagation conditions for an industrially used Bacillus licheniformis, this study examines the effect of transferring cells at the early-stationary growth phase (pH 5.3) to fresh growth medium at pH 5.0-8.0. Intracellular pH (pHi) was measured on a single-cell level, using fluorescence ratio imaging microscopy after staining with 5(6)- carboxyfluorescein diacetate succinimidyl ester. Transcription profiles were determined using a genome DNA microarray. The optimum extracellular pH (pH ex) value for growth of B. licheniformis was found to be pH 7.0, resulting in the shortest lag phase, highest maximum specific growth rate and maximum biomass formation. An average pH gradient (ΔpH = pHi - pHex) of approx. 1.0 was found in B. licheniformis 15 min after transfer to pHex 5.0-8.0. Up-regulation of genes involved in sucrose uptake at pH 7.0 could be related to the optimum growth observed. Transcription profiles indicated that the organism was experiencing phosphate starvation upon transfer to pH 7.0 and pH 8.0. Mechanisms involved in pHi regulation appeared to include changes in fatty acid synthesis to yield a more rigid cell membrane structure at low pHex values and conversion of pyruvate to acetoin instead of acetate for neutralization of low pHex values. © Springer-Verlag 2004.

Raghevendran, V., Gombert, A.K., Christensen, B., Kötter, P., Nielsen, J.

"Phenotypic characterization of glucose repression mutants of Saccharomyces cerevisiae using experiments with 13 C-labelled glucose"

Yeast, 21 (9), pp. 769-779. (2004)

In the field of metabolic engineering and functional genomics, methods for analysis of metabolic fluxes in the cell are attractive as they give an overview of the phenotypic response of the cells at the level of the active metabolic network. This is unlike several other high-throughput experimental techniques, which do not provide information about the integrated response a specific genetic modification has on the cellular function. In this study we have performed phenotypic characterization of several mutants of the yeast Saccharomyces cerevisiae through the use of experiments with 13C-labelled glucose. Through GC-MS analysis of the 13C incorporated into the amino acids of cellular proteins, it was possible to obtain quantitative information on the function of the central carbon metabolism in the different mutants. Traditionally, such labelling data have been used to quantify metabolic fluxes through the use of a suitable mathematical model, but here we show that the raw labelling data may also be used directly for phenotypic characterization of different mutant strains. Different glucose derepressed strains investigated employed are the disruption mutants reg1, hxk2, grr1, mig1 and mig1mig2 and the reference strain CEN.PK113-7D. Principal components analysis of the summed fractional labelling data show that deleting the genes HXK2 and GRR1 results in similar phenotype at the fluxome level, with a partial alleviation of glucose repression on the respiratory metabolism. Furthermore, deletion of the genes MIG1, MIG1/MIG2 and REG1 did not result in a significant change in the phenotype at the fluxome level. Copyright © 2004 John Wiley & Sons, Ltd.

Connelly, M.B., Young, G.M., Sloma, A.

"Extracellular proteolytic activity plays a central role in swarming motility in Bacillus subtilis"

Journal of Bacteriology, 186 (13), pp. 4159-4167. (2004)

Natural isolates of Bacillus subtilis exhibit a robust multicellular behavior known as swarming. A form of motility, swarming is characterized by a rapid, coordinated progression of a bacterial population across a surface. As a collective bacterial process, swarming is often associated with biofilm formation and has been linked to virulence factor expression in pathogenic bacteria. While the swarming phenotype has been well documented for Bacillus species, an understanding of the molecular mechanisms responsible remains largely isolated to gram-negative bacteria. To better understand how swarming is controlled in members of the genus Bacillus, we investigated the effect of a series of gene deletions on swarm motility. Our analysis revealed that a strain deficient for the production of surfactin and extracellular proteolytic activity did not swarm or form biofilm. While it is known that surfactin, a lipoprotein surfactant, functions in swarming motility by reducing surface tension, this is the first report demonstrating that general extracellular protease activity also has an important function. These results not only help to define the factors involved in eliciting swarm migration but support the idea that swarming and biofilm formation may have overlapping control mechanisms.

Hornbæk, T., Nielsen, A.K., Dynesen, J., Jakobsen, M.

"The effect of inoculum age and solid versus liquid propagation on inoculum quality of an industrial Bacillus licheniformis strain"

FEMS Microbiology Letters, 236 (1), pp. 145-151. (2004)

Shorter lag phases were obtained in cultivations of Bacillus licheniformis using early-compared to late-stationary growth phase inocula and using liquid versus solid propagation medium. Flow cytometry and fluorescence ratio imaging microscopy (FRIM) after staining with 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), confirmed that liquid early-stationary growth phase inoculum had a higher vitality and was more homogeneous than solid late-stationary growth phase inoculum. DNA-microarray analyses indicated that liquid early-stationary growth phase inoculum was in a more active state in terms of cell multiplication whereas solid late-stationary growth phase inoculum was induced to some spore formation potentially causing delayed growth initiation. © 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

S. Bhargava; K. Wenger; M. R. Marten.

"Pulsed feeding during fed-batch Aspergillus oryzae fermentation leads to improved oxygen mass transfer."

Biotechnololy Prog. 19, 1091-1094 (2003)

Productivity in many fungal fermentations is detrimentally affected by high broth viscosity and consequent reduced oxygen mass transfer capacity. The goal here was to determine whether pulsed feeding of limiting carbon in a fungal fermentation could lead to reduced viscosity and improved oxygen mass transfer. Compared to continuous feeding, pulsed addition of substrate led to smaller fungal elements, which resulted in a significant reduction in broth viscosity. This in turn led to higher dissolved oxygen concentrations and increased oxygen uptake rates during pulsed feeding.