Comparison of four different process spectrocopy instruments in the evaluation
of a Bioethanol production process
The use of process analytical technology (PAT) for bioethanol production is an exciting and potentially very powerful development. Many spectroscopic techniques rely on the vibrational spectroscopies: Fourier transform mid infrared (FT-mid-IR), Raman spectroscopy and near infrared (NIR). Different techniques have their own strengths and weaknesses but are seldom compared to established which really is the best techniques for a given application.
Keit has developed an innovative FT-mid-IR spectrometer that contains no moving parts, and removes the need for fragile fibre probes, cooling engines or liquid nitrogen that is required by conventional FT-mid-IR instruments.
Here we compare the Keit IRmadillo directly with conventional process FT-mid-IR, Raman and NIR spectrometers for in situ monitoring of a bioethanol production process. A fermentation using S. cerevisiae as the organism and sucrose as the feedstock was performed in a 25 L stainless steel Applikon fermenter, with the comparison spectrometer probes inserted through standard ports.
Figure 2 shows the concentration prediction for ethanol produced over the course of the fermentation. The reference values shown on the graph are derived from off-line HPLC, and are the same in all cases.
IRmadillo FT-mid-IR: showed the best correlation with the reference points, with small error bars and a clear concentration trace the passes through all of the reference points, including those immediately before and after the sucrose feed.
Conventional FT-mid-IR: the conventional FT-mid-IR tracked the initial production of ethanol, but then struggled with the higher concentrations.
Raman: this instrument performed in a similar way to the IRmadillo, but the error bars were larger with greater variation from sample to sample.
NIR: this instrument showed the correct general trend, but was less accurate with a consistent offset on the prediction, over-predicting the ethanol concentration.
Figure 3 shows the concentration prediction for the initial sucrose feedstock, as well as glucose and fructose.
IRmadillo FT-mid-IR: this was the only instrument that did not predict negative concentrations for sucrose. The concentration curve for sucrose is clear and correlates extremely well with the reference points. The glucose and fructose measurements are also clearly monitored, including the decrease in all sugar concentrations before the sucrose feed, as well as the rapid increase with feeding.
Conventional FT-mid-IR: this did predict the general trend for sucrose, but with a high degree of error and did not accurately measure at low concentrations, showing a negative concentration. The general trend for glucose was measured, but the fructose concentration was not predicted accurately.
Raman: the Raman did not accurately monitor the concentration of sucrose, with notable divergence from the reference values. This was especially noticeable after the feed, where a high biomass concentration was evident. It is likely that fluorescence strongly affected the measurement here The fructose and glucose concentrations were similar to the IRmadillo predictions.
NIR: the NIR shows a reasonable, yet noisy prediction for sucrose in the early stages of the fermentation, but struggles post sucrose feed.
The glucose predictions are fairly accurate, but the NIR cannot monitor fructose prediction at all.
These results shows that the IRmadillo is not only equivalent to alternative conventional process spectroscopies, but is arguably the most accurate technique available.
The improved performance over conventional FT-mid-IR comes from the robustness of the solid state design, meaning no moving mirrors or fibre optics are required. This means that spectral quality is improved, with reliable and stable spectra.
The improvement over Raman comes from the simple design and principals of FT-mid-IR compared to Raman, meaning that both fluorescence and scattering effects can safely be ignored.
The improvement over NIR comes from the fact that FT-mid-IR spectroscopy directly measures the features of interest, rather than overtones and combination bands. This means similar molecules (such as comparing one molecule of sucrose with one molecule of fructose and glucose) can be identified and quantified much easier than with NIR.
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