The scale-up of microbial batch and fed-batch fermentation processes

Micro-organisms are important for both human health and to industry so the fed-batch cultivation of microbial strains, often over expressing recombinant or natural proteins, to high cell density has become an increasingly important technique throughout the field of biotechnology, from basic research programmes to large-scale pharmaceutical production processes (Hewitt et al., 1999). The scale-up of such a process is usually the final step in any research and development programme leading to the large-scale industrial manufacture of such products by fermentation (Einsele, 1978). It is important to understand that the process of scaling-up a fermentation system is frequently governed by a number of important engineering considerations and not simply a matter of increasing culture and vessel volume. Therefore, it is perhaps surprising when the large-scale does not perform as well as the small-scale laboratory process. It is often observed that the biomass yield and any growth associated products are often decreased on the scale-up of an aerobic process (Enfors et al., 2001). For Saccharomyces cerevisiae, the biomass yield on molasses increased by 7% when the process was scaled-down from 120 m3 to 10 l even when a seemingly identical strain, medium and process were employed (George et al., 1993). In an E. coli fed-batch recombinant protein process, the maximum cell density reached was found to be 20% lower when scaling-up from 3l to 9 m3 and the pattern of acetic acid formation had changed. (Bylund et al., 1998). During another study (Enfors et al., 2001), the performance of a recombinant strain of E. coli during fed-batch culture was found to vary on scale-up from the lab-scale to 10-30 m3 industrial bioreactors. This included lower biomass yields, recombinant protein accumulation and surprisingly perhaps a higher cell viability. These findings are typical of those found when scaling up most fermentation processes yet only a few mechanisms have been presented that can satisfactorily explain these phenomena. In this Chapter, we will briefly discuss the main engineering considerations involved in fermentation scale-up and then critically review those mechanisms thought to be responsible for any detrimental change in bioprocessing at the largerscale. Though it addresses mainly E. coli fed-batch fermentations, much of the discussion also applies to batch and other single celled aerobic microbial fermentations too

Formulation optimisation of mixed sugar/protein/maltodextrin encapsulants for spray drying L. acidophilus using the response surface method

Three sugars (maltose, fructose, and lactose) have been combined in different formulations with three protein based powders (whey protein, skim milk, and soy protein) to assess the survivability of L. acidophilus after spray drying at 80°C followed by optional further exposure to simulated gastric intestinal juice (SGI) or bile solution. The results showed that the highest survival rate was found in a recipe consisting of 87.5% skim milk and 12.5% maltose, while the lowest rates were found in formulations containing no protein. Maltose and lactose provide higher survival rate than fructose which may reflect the higher glass transition temperature of maltose/lactose mixtures. Similar trends were found with cells rehydrated in SGI and bile solutions

A comparison of the survival rates of E. coli K12 and L. acidophilus in spray drying

The survival of mid-exponential and the early-stationary E. coli K12 and L. acidophilus were investigated when spray drying and outlet air temperatures of 60, 70, 80, 90 and 100°C. The results showed that the early-stationary cell of both cultures had a greater heat resistance than the mid-log cell in every drying temperature. The best survival rate was found when spray drying at temperature lower 80°C and it is showed that L. acidophilus is stronger than E. coli K12 (irrespective of the growth phase)

Studies supporting the use of mechanical mixing in large scale beer fermentations

Brewing fermentations have traditionally been undertaken without the use of mechanical agitation, with mixing being provided only by the fluid motion induced by the CO2 evolved during the batch process. This approach has largely been maintained because of the belief in industry that rotating agitators would damage the yeast. Recent studies have questioned this view. At the bench scale, brewer’s yeast is very robust and withstands intense mechanical agitation under aerobic conditions without observable damage as measured by flow cytometry and other parameters. Much less intense mechanical agitation also decreases batch fermentation time for anaerobic beer production by about 25% compared to mixing by CO2 evolution alone with a small change in the concentration of the different flavour compounds. These changes probably arise for two reasons. Firstly, the agitation increases the relative velocity and the area of contact between the cells and the wort, thereby enhancing the rate of mass transfer to and from the cells. Secondly, the agitation eliminates spatial variations in both yeast concentration and temperature, thus ensuring that the cells are maintained close to the optimum temperature profile during the whole of the fermentation time. These bench scale studies have recently been supported by results at the commercial scale from mixing by an impeller or by a rotary jet head, giving more consistent production without changes in final flavour. It is suggested that this reluctance of the brewing industry to use (adequate) mechanical agitation is another example where the myth of shear damage has had a detrimental effect on the optimal operation of commercial bioprocessing

Multiparameter flow cytometry for the characterization of human embryonic stem cells

Using multiparameter staining methods and flow cytometry to investigate the pluripotency of HUES7 human embryonic stem cell cultures, it was found that the multidimensional approach of marker co-expression allowed the different cell populations to be easily identified and demonstrated cross reactivity between the SSEA 4 and SSEA 1 antibodies, resulting in a substantial false positive SSEA 1 population. It is the accepted norm to apply control gates at a 95 % confidence level of the isotype control; however, this study found that adjusting the control gate to a 99 % confidence level significantly reduced the effect of this cross reactivity. Though conversely, this gating shift also decreased the positive marker expression of SSEA 4 and Tra-1-60, indicating that there is a need for strongly expressing markers coupled with increased optimization of fluorophore/antibody combinations before a gating strategy of 99 % can be implemented on a more routine basis

Scale‐down studies for the scale‐up of a recombinant Corynebacterium glutamicum fed‐batch fermentation; loss of homogeneity leads to lower levels of cadaverine production

BACKGROUNDThe loss of efficiency and performance of bioprocesses on scale‐up is well known, but not fully understood. This work addresses this problem, by studying the effect of some fermentation gradients (pH, glucose and oxygen) that occur at the larger scale in a bench‐scale two‐compartment reactor (Plug flow reactor (PFR) + Stirred tank reactor (STR)) using the cadaverine‐producing recombinant Corynebacterium glutamicum DM1945 Δact3 Ptuf‐ldcC_OPT. The new scale‐down strategy developed here studied the effect of increasing the magnitude of fermentation gradients by considering not only the average cell residence time in the PFR (τPFR), but also the mean frequency at which the bacterial cells entered the PFR (fm) section of the two‐compartment reactor.RESULTSOn implementing this strategy the cadaverine production decreased on average by 26 %, 49 % and 59 % when the τPFR was increased from 1 min to 2 min and then 5 min respectively compared to the control fermentation. The CO2 productivity was highest (3.1‐fold that of the control) at a τPFR of 5 min, but no losses were observed in biomass production. However, the population of viable but non‐culturable cells increased as the magnitude of fermentation gradients was increased.CONCLUSIONThis study demonstrated that C. glutamicum DM1945 Δact3 Ptuf‐ldcC_OPT physiological response was a function of the magnitude of fermentation gradients simulated. The adaptations of a bacterial cell within a heterogeneous environment ultimately result in losses in fermentation productivity as observed here.</div

Multiparameter flow cytometry for the characterisation of extracellular markers on human mesenchymal stem cells

Extracellular surface proteins are used to identify fully-functional human mesenchymal stem cells (hMSCs) in a mixed population. Here, a multiparameter flow cytometry assay was developed to examine the expression of several bone marrow-derived hMSC markers simultaneously at the single cell level. The multiparameter approach demonstrates a depth of analysis that goes far beyond the conventional single or dual staining methods. CD73, CD90 and CD105 were chosen as positive markers as they are expressed on multipotent hMSCs, whilst CD34 and HLA-DR were chosen as negative indicators. Single colour analysis suggested a population purity of 100 %; in contrast, when analysed via the multiparameter method, the CD73/CD105/CD90/HLA-DR/CD34 phenotypes represented 94.5 ± 1.3 % of the total cell population. Also, although CD271 has been posited as a definite early stage hMSC marker, here we show it is not present on pre-passage cells, highlighting the need for careful marker selection. © 2013 Springer Science+Business Media Dordrecht

A quantitative approach for understanding small-scale human mesenchymal stem cell culture implications for large-scale bioprocess development

Human mesenchymal stem cell (hMSC) therapies have the potential to revolutionise the healthcare industry and replicate the success of the therapeutic protein industry; however, for this to be achieved there is a need to apply key bioprocessing engineering principles and adopt a quantitative approach for large-scale reproducible hMSC bioprocess development. Here we provide a quantitative analysis of the changes in concentration of glucose, lactate and ammonium with time during hMSC monolayer culture over 4 passages, under 100% and 20% dissolved oxgen (dO2), where either a 100%, 50% or 0% growth medium exchange was performed after 72h in culture. Yield coefficients, specific growth rates (h-1) and doubling times (h) were calculated for all cases. The 100% dO2 flasks outperformed the 20% dO2 flasks with respect to cumulative cell number, with the latter consuming more glucose and producing more lactate and ammonium. Furthermore, the 100% and 50% medium exchange conditions resulted in similar cumulative cell numbers, whilst the 0% conditions were significantly lower. Cell immunophenotype and multipotency were not affected by the experimental culture conditions. This study demonstrates the importance of determining optimal culture conditions for hMSC expansion and highlights a potential cost savings from only making a 50% medium exchange, which may prove significant for large-scale bioprocessing

An investigation into the preservation of microbial cell banks for alpha-amylase production during 5L fed-batch Bacillus licheniformis fermentations

Fluorescent staining techniques were used for a systematic examination of methods used to cryopreserve microbial cell banks. The aim of cryopreservation here is to ensure subsequent reproducible fermentation performance rather than just post thaw viability. Bacillus licheniformis cell physiology post-thaw is dependent on the cryopreservant (either Tween 80, glycerol or dimethyl sulphoxide) and whilst this had a profound effect on the length of the lag phase, during subsequent 5 l fed-batch fermentations, it had little effect on maximum specific growth rate, final biomass concentration or a-amylase activity. Tween 80 not only protected the cells during freezing but also helped them recover post-thaw resulting in shorter process times

Physiological effects of the addition of n-dodecane as an oxygen vector during steady-state Bacillus licheniformis thermophillic fermentations perturbed by a starvation period or a glucose pulse

The effect of the presence of n-dodecane as a potential oxygen vector during oxygen limited continuous cultures of a Bacillus strain was studied, under extreme nutrient supply conditions: glucose excess, limitation and starvation. The addition of n-dodecane to the aqueous phase of a mechanically agitated and aerated fermentation increased the kLa by up to 35%. The n-dodecane additions to B. licheniformis cells during starvation (oxygen limitation with concomitant glucose starvation) caused a severe detrimental progressive change in cell physiological state with respect to cytoplasmic membrane polarisation and permeability which was mitigated against by alleviating either the oxygen limitation (by increasing the mean energy dissipation rate or by the addition of n-dodecane as an oxygen vector) or by alleviating the carbon limitation (by resuming the carbon feed or by the addition of a glucose pulse). Further that during periods of excess glucose (glucose pulse) a much higher kLa was required to prevent the onset of anaerobic mixed acid fermentation than could be provided by the addition of n-dodecane alone. N-dodecane can be used to increase the kLa when added in sufficient quantities to the aqueous phase of a mechanically agitated and aerated bioreactor but the magnitude of this increase is process and vessel geometry specific