An identical peristaltic pump pushed deionized water through the flow cell. The in-line sampling protocol was as follows: 1.5 min of flushing the optical cell with water, 1.5 min with water “reference” measurement, 1.5 min of wine flowing through the cell, and 1.5 min of sample measurement. A total of 100 mL of wine is shunted during the 3-min pump time. The water and wine flow is halted during the absorbance measurements to avoid bubble contamination. The peristaltic pumps were controlled using LabView via a solid-state AC relay and a digital interface . Control samples were also manually drawn coinciding with the automatic sample times, placed in 50 mL plastic tubes and immediately stored in a refrigerator at -4°C for later analysis.The performance of the LED colorimeter was validated using an in-line and laboratory experimental configuration. The in-line study examines the effect of interference from residual particles in the wine solution after a 2.0 µm filtration. Samples were manually drawn at the in-line sampling time and centrifuged offline before being analyzed using the LED colorimeter. The study quantifies the absorbance offset contribution from particles by comparing measurements made on red wine fermentation samples using either the 2.0 µm filter or centrifuge. A second investigation was used to determine if the LED colorimeter could be used as a replacement for a UV-vis spectrophotometer. A large number of fermentation samples were collected and measured using the LED colorimeter and UV-vis after a centrifuge step. A strong correlation was found between absorbance measurements made with the LED sensor and UV-vis spectrophotometer for total phenolic and color.

A phenolic extraction plot comparing in-line and laboratory absorbance measurements during fermentation is shown in Figure 3. The phenolic measurements shown are made with the LED colorimeter instrument. The in-line method used a pump to push fermentation samples through a filter after which total phenols and red color were measured and recorded. The laboratory method used a centrifuge on samples that were manually collected at the in-line sampling times. The absorbance measurements for the in-line method were always greater than the laboratory centrifuge system. The absorbance offset is most pronounced for the total phenolic measurement with the background offset of the in-line method approaching 50% of the total absorbance measured. In contrast,gardening pots plastic the color measurement had a background offset of at most 25% of the total absorbance. The background offset arises from suspended particles in the sample. Light is scattered by these particles, leading to an effective background absorbance. The in-line absorbance offset limits the dynamic range of the measurement and obscures the true absorbance value. Standard analytical sample preparation removes essentially all particles above a dimension of 0.45 µm. The 0.45 µm membrane filters clog frequently while the 2.0 µm filtration reduces the particle level to a tolerable level in the background absorbance. The measurement focus of this study was to validate if the LED sensor is a suitable replacement for a UV-vis spectrometer in making these absorbance measurements. A plot comparing absorbance measured using the LED sensor and UV-vis for a single Shiraz fermentation is shown in Figure 4. A total of 28 samples were collected during fermentation, of which two were excluded from this analysis because of interference from carbon dioxide bubbles. The absorbance measured using the LED sensor for the wavelength 280 nm is smaller than the UV-vis measurement for all samples.

The opposite trend was found for the 420 nm LED with the UV-vis measurement now smaller. The color is approximately equal to the UV-vis measurement. These results demonstrate that absorbance measurements made with LED sources are closely related to the spectral region of the measurement. The broad spectral bandwidth of LED sources results in more or less light in the measurement compared to a narrow bandwidth UVvis measurement. Several groups developed a model to relate the effect of broad bandwidth LED sources on absorbance measurements and used it to study a variety of chemical analytes. Smith and Cantrell found that the best linearity is achieved with narrow LED sources centered on broad spectral absorbance features. To test if the relationship between LED and UV-vis absorbance was systematic, a correlation study was used on all fermentation samples collected during the Fall 2012 season . Figure 5 shows a correlation plot comparing LED and UV-vis absorbance measurements for wavelengths 280, 420, and 525 nm. The absorbance measurements made with LEDs showed high correlation for both total phenols and red color , but performed only adequately for the 420 nm and other wavelengths. A plot showing phenolic extraction from UV-vis absorbance measurements of days 1, 3, and 6 of a red wine fermentation is shown in Figure 6. The slope and shape of the absorbance feature changes very little for the total phenols and red color measures with regard to time of extraction. In contrast, the 420 nm and 630 nm shape of the absorbance feature changes substantially over the six fermentation days shown. The poor correlation observed is the result of making a measurement over this non-static absorbance region with a broad spectral bandwidth LED. This is consistent with previous reports and indicates that LED absorbance measurements are dependent on the overlap of LED and absorbance spectrums. The total phenols and red color absorbance measurements made using the LED sensor were reduced by 40% and 10% respectively, compared to the UV-vis measurement. The high correlations between the two methods for the total phenols and red color allow simple corrections to be applied, translating the absorbance into an equivalent UV-vis measurement.

The phenols extracted during red wine fermentation are strongly related to the fermentation temperature. Three manually sampled fermentations of the same Shiraz fruit were fermented at temperatures of 15, 20, and 25 ºC. The absorbance measurements for total phenols and red color are shown in Figure 7 for a 16-day fermentation. The rate of total phenol and red color extraction is greater when the fermentation temperature is larger. The absorbance approaches a steady state saturation value for both total phenols and red color, suggesting that the rate, but not saturation value, is temperature dependent. The challenges encountered in developing an in-line absorbance sensor can be divided between opto-electronic and fluidic delivery issues. The opto-electronic aspect of the design required a miniaturized jig with minimal distance from LED to photo diode so as to optimize the delivery and capture of photons on the detector surface. Optimizing the photon flux onto the detector surface improves the signal to noise ratio, allowing larger optical densities to be measured. One challenge in using UV LEDs as optical sources is optimizing the light collection on the photo diode detector. The optical power of the UV LED sources is very low and matters are further complicated by the poor responsitivity of the photo diodes at the UV wavelengths. The collection efficiency of the LED sensor was optimized by using large area photo diodes and minimizing the distance between LED and the detectors. The collection efficiency was good with this distance being fixed at 2.6 mm, the thickness of the flow cell. The maximum photo current for the 280 nm LED was measured at 2 μA. Silicon photo diodes with UV transparent windows have a low responsivity of 0.13 A/W at a wavelength of 280 nm. Even under the most optimistic circumstances, in which 100% photon coupling is achieved with no reflective losses, the photo current would only be 65 μA. The existing design using a ball lens on the UV LED sources captures nearly all of the light available,plastic pots with drainage holes as the UV LED spot size is much smaller than the UV photo diode diameter. An alternative approach is to forego the focusing ball lens and to instead use a flat window. The approach requires a large area photo diode and a very short path length between source LED and photo diode. A simple calculation shows that a flat window UV LED with 60-degree view angle and UV photo diode with 2.54 mm diameter would require a path length of 0.7 mm to capture all light. The short path length constraint adds complexity, requiring a customized optical flow cell. A ball lens is required in the current LED sensor because the thickness of the flow cell is 2.6 mm. Future investigations might focus on miniaturized flow cells, where the ball lens is removed to reduce overall system cost. In chemical analysis, a standard sample preparation is to filter samples using 0.45 µm to remove all suspended particles and microorganisms. Using a 0.45 µm filter for in-line sample clarification of must is difficult, with frequent clogging as yeast and pulp accumulate on the membrane surface. Offset correction schemes using reference wavelengths to monitor the extent of optical scattering have been adapted for in-line processes . The 630 nm LED used in this sensor did not successfully quantify the optical scattering in these red wine fermentation measurements. As shown in Figure 4, the change in absorbance of 0.75 AU for the 630 nm LED wavelength is not in the instruments’ noise, and represents a real change in the measured absorbance of roughly 1% transmittance. This 1% change in transmittance is likely attributed to the shoulder of the color absorbance. Another option would be the monitor at a reference wavelength further in the near infrared region for an estimate of the optical scattering. In this region, absorbance is essentially independent of phenol or color contributions. The use of background correction schemes using reference wavelength absorbance measurements is advocated because it might reduce the in-line filtration requirement. This is important because it will ensure filter life during each fermentation. A 2.0 µm pore filter in a 47 mm housing was used to remove gross particulates from the sampled wine, and effectively removed the major portion of this background absorbance contribution.

The problem of carbon dioxide release leading to bubble formation in the flow cell was also addressed by the in-line filtration under pressure of the peristaltic pump. Bubbles can obscure the path length and lead to random variation in the measured absorbance. No additional approach was made in the current experimental setup to avoid or remove bubbles. One approach described for HPLC-based capillary system was sufficient back pressure to avoid out gassing and bubble formation . Another approach might be to adopt an open-cell dip-type reflectance probe where the sample could be agitated to remove bubbles. Bubble formation is most prevalent ~12 to 36 hr after inoculation of the fermentation, when the Brix begins dropping most rapidly. To address bubble interference in the in-line trials, the measurements were repeated until consistent results were achieved. In repeating the measurement, the wine pump was turned on to flush the cell with a new sample. Sample measurements are judged to be of poor quality when the standard deviation for any LED wavelength is poor . The primary concern in repeating sample measurements is that it requires frequent replacement of the filter due to a build-up of particles on the membrane surface. Reducing the sample volume or the sampling frequency is critical for prolonging the membrane filter for in-line measurements. Future studies might investigate minimizing the volume of wine that flows across the filter to ensure its lifetime during fermentation.However, the application of these technologies has been hampered by public apprehension toward potential food safety and gene flow concerns, resulting from the presence and/or expression of transgenes. Recent development of CRISPR/Cas9-mediated genome editing has made targeted mutagenesis an attractive alternative to traditional transgenic technologies . In plants, the most widespread application of CRISPR/Cas9 entails the stable integration of Cas9 endonuclease and single-guide RNA genes into host genomes. When CRISPR/Cas9-mediated gene editing is used in sexually propagated annual crop plants, transgenes can be eliminated from host genomes following sexual reproduction and screening of segregating populations. This segregation of CRISPR/Cas9 transgenes from mutations of interest can result in non-transgenic mutant plant progeny. However, this strategy is rarely feasible or practical for vegetatively propagated perennial plants. These plants generally require years to reach sexual maturity; thus, multiple years are needed before sexual reproduction is feasible. Additionally, these plants are highly heterozygous for genes controlling many important traits, and these traits will segregate and recombine following sexual reproduction, resulting in non-transgenic mutant progeny likely lacking a combination of desirable traits. Developing a method to generate CRISPR/Cas9-mediated non-transgenic mutants is highly desirable for many applications of genome editing, particularly for asexually propagated, heterozygous, perennial crop plants.