26 research outputs found
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, brewerâs
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
Scaleâdown studies for the scaleâup of a recombinant Corynebacterium glutamicum fedâbatch fermentation; loss of homogeneity leads to lower levels of cadaverine production
BACKGROUNDThe loss of efficiency and performance of bioprocesses on scaleâup 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 benchâscale twoâcompartment reactor (Plug flow reactor (PFR) + Stirred tank reactor (STR)) using the cadaverineâproducing recombinant Corynebacterium glutamicum DM1945 Îact3 PtufâldcC_OPT. The new scaleâdown 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 (ÏPFR), but also the mean frequency at which the bacterial cells entered the PFR (fm) section of the twoâcompartment reactor.RESULTSOn implementing this strategy the cadaverine production decreased on average by 26 %, 49 % and 59 % when the ÏPFR was increased from 1 min to 2 min and then 5 min respectively compared to the control fermentation. The CO2 productivity was highest (3.1âfold that of the control) at a ÏPFR of 5 min, but no losses were observed in biomass production. However, the population of viable but nonâculturable cells increased as the magnitude of fermentation gradients was increased.CONCLUSIONThis study demonstrated that C. glutamicum DM1945 Îact3 PtufâldcC_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