NIR Microspectroscopy to Determine the Composition of Visualised Droplets

Using microscopic observation of fermentation broth from both the M4018 and M3 8182 processes it was possible to visually distinguish droplets. On dying the oil fed to each of these processes, dyed droplets were observed. This strongly suggested that these

droplets were composed of oil. To eliminate the possibility that these droplets were composed of anything other than oil, for example bubbles of air, near infra-red (NIR) microspectroscopy was used. This allows determination of a particular scanned substance in a mixture by comparison with reference scans of the individual components.

6.2.1. NIR Scanning of Pure Samples of the Major Media Components

Initially pure samples of the major media constituents were scanned. These were RSO, soybean meal and dried blood meal. Since oil containing SbB dye was used in the visualisation fermentations, oil containing 3 %(w/w) SbB dye was also scanned. The results of these individual scans are grouped and shown in Figure 6.11.

c o H. o C/5 X) 0.6 0.4 0.2 0.0 -0.2 -0.4 -0.6 •0.8 1600 1700 1800 1900 2000 2100 2200 2300 2400 Wavelength (nm)

Figure 6.11. Absorption profile of individual samples of dried blood meal ( ), soybean meal ( ), rape seed oil ( ) and rape seed oil containing 3 %(w/w) SbB (—). The scan was performed as described in Section 2.2.2.6.

C h apter 6. Visualisation o f The D istribution a n d Size o f Residual O il D roplets

The scan of RSO (Figure 6.11.) showed two regions of distinct peaks/troughs at around 1650-1750nm and 2250-2350nm. There was also a weaker peak at 2152nm. The

addition of 3 %(w/w) SbB had no significant affect on the absorbance profile of the RSO (Figure 6.11.). The scans of the dried blood meal and soybean meal showed several common regions of absorbance (Figure 6.11.). The peaks/troughs in the 1800 to 2000nm region can be disregarded as these are due to water. There are also distinctive peaks/troughs in the 2000 to 2200nm region. Other peaks/troughs of interest were those at 1650-1750nm and 2250-2350nm, as these were similar to those observed in scans of RSO. However, when scans of the soybean and blood meals were compared to that from RSO, the peaks/troughs observed in the 2250-2350nm region displayed a shift to lower wavelength (Figure 6.11.). This shift is in fact significant enough to distinguish between RSO and the soybean and blood meals {Personal communication, Hammond, 1998). In addition, the soya and blood meals can be further distinguished from oil due to the peaks/troughs between 2000nm and 2200nm. Furthermore in the RSO scan, the ‘Veak peak” observed at 2152nm was unique. Using these characteristic features, it should be possible to identify if a droplet scanned in a fermentation broth sample is indeed composed of oil.

6.2.2. NIR Scanning of the Microscopic Droplets in the Broth

Although NIR radiation can transmit through glass, infra-red radiation cannot. Consequently the lens of the microscope component of these systems is historically composed of a series of mirrors, or Cassegrain lens (Blanco, 1998). As a result of having a mirror based lens energy is lost. Thus under similar levels o f magnification, the clarity and resolution of the observed image is of lower quality to that obtained from a traditional light microscope. A consequence of this was the inability to resolve

unstained oil droplets with the NIR system microscope. Figure 6.12. however, shows a typical image of a sample of broth from a M4018 fermentation which had been fed with oil containing 3 %(w/w) SbB. It was, therefore, possible to distinguish stained droplets, and NIR absorption scans were carried out on many o f these. Figure 6.13 shows an example of a scan of one of these droplets. This scan shows that the droplets had an absorbance profile very similar to that of RSO. The exception to this was the

peaks/troughs between 1800nm and 2000nm, which were due to the presence of water. Water was apparent due to its presence in the fermentation broth surrounding and coating the droplet. The presence of the surrounding broth also accounts for the weaker response when compared to that of the pure oil samples. Indeed, the weak peak at

2 1 52nm becam e too w eak to be significant in the scans o f droplets w ithin the broth. N IR scans o f several droplets in sam ples taken at different tim e points from an M 4018 ferm entation fed w ith 3 % (w /w ) SbB w ere plotted w ith scans o f the reference m aterials (Figure 6.14.). It is apparent that peaks/troughs at 2250nm and 2302nm coincided in both the RSO reference (w ith and w ithout dye addition) and droplet scans. Furtherm ore, the droplet scans did not show absorption peaks/troughs in the 2000nm to 2200nm w avelength regions characteristic to the soybean and blood m eals. T his is strong evidence to confirm that the droplets observed w ithin the broth w ere indeed com posed o f oil.

Stained droplets

F ig u re 6.12. M icr o g ra p h o f b ro th sa m p le tak en at 113 h o u r s fro m an M 4 0 1 8 fe r m e n ta tio n fed w ith oil c o n ta in in g 3 % (w /w ) S b B , as v ie w e d w ith th e N IR m icr o sco p e. S lid e p rep a re d a c c o r d in g to sectio n 2 .2 .2 .6 .2 .

0.6 0.4 0.2 c 2 0.0 ^ -0.2 < -0.4 -0.6 1600 1700 1800 1900 2000 2100 2200 2300 2400 W avelength (nm)

F ig u re 6 .13 . T y p ic a l N IR a b so r p tio n p ro file o f a d y ed d r o p le t in M 4 0 1 8 p ro cess b ro th . S a m p le p rep a re d a c c o r d in g to S ectio n 2 .2 .2 .6 2 . a n d a n a ly se d as d e sc r ib e d in S ectio n 2 .2 .2 .6 .3 .

Chapter 6. Visualisation o f The Distribution and Size o f Residual O il Droplets