## 3 Results and Discussion
### Validation of the New Method  
Reproducibility of the coated QCM sensor was evaluated by the repeated measurements of anaerobic digestate sample (day 42) using the single QCM disc; the obtained results are presented in Figure 6. According to the researchers, RSD should not exceed 20% [34]. In this case, as shown in Figure 6, there is a slight variation among the values of frequency-shift rates and the RSD is 13.6%, which is within the proper range. However this RSD value is higher than those obtained using GC by [PERSON] et al. [34]. In terms of accuracy, GC analysis seems to perform better. The variation of frequency-shift rates is likely due to the manual-based coating method in this study because the QCM disc is newly coated every time. Actual, the manually coated thin film does not look uniform; thus a coating equipment should be used to improve reproducibility.  
It has been shown by [PERSON] et al. [35] that the inhibition is more closely related to the LCFAs adsorbed onto microbial cells (so called biomass-associated LCFAs) than the free LCFAs in liquid phase. Therefore it is more useful for AD to measure biomass-associated LCFAs with the QCM sensor. However, it is still unclear if the present pretreatment method including pH adjustment and heating can elute the biomass-associated LCFAs into alkaline water. An additional series of experiments was carried out to understand the distribution ratio of LCFAs between water and solid (biomass). Table 2 summarizes LCFA recoveries in the pretreatment using three different digestate samples. The results indicate that the pretreatment method used in this study cannot recover biomass-associated LCFAs; namely the QCM sensor might measure free LCFAs in liquid phase. The LCFAs in the sediment accounts for 36%-58% of the total LCFAs in the sludge and the mass concentrations in 1 g TSS are 23 mg/g-TSS (Sludge A), 25 mg/g-TSS (Sludge B) and 49 mg/g-TSS (Sludge C), respectively. Sludge C, which was strongly inhibited by LCFAs, accumulates more LCFAs in biomass. Even so, it seems that similar increasing trends can be obtained between total LCFA concentrations and frequency-shift rates since LCFAs in supernatant is increased with total LCFA concentrations in sludge as shown in Table 2. To improve recovery of biomass-associated LCFAs, the modified pretreatment method should be designed. [PERSON] et al. [34] successfully extracted LCFAs in organic phase from solid samples. Since dissociation elutes LCFAs in organic solvent into water, it might be better that the pH adjustment follows the first extraction using organic solvent.  
Figure 6: Reproducibility of frequency-shift rates from the same QCM sensor (\(n=7\)).  
From the above discussion, it was found that the proposed method is less accurate in terms of reproducibility than GC analysis. The conventional GC analysis should be used to know more scientifically reliable and detailed LCFA data. Nevertheless the proposed method offers the advantage of providing results by a simple device in a shorter time as the overall experiment, including the pretreatment for each sample, takes only 1 h. A QCM sensor is considered a cost-effective device [36], whereas consumable costs depend on a costly quartz disc. The above-mentioned result indicates that the simple coating method enables the regeneration of sensor response, which contributes to reducing the cost. Previously, it has been reported that a QCM sensor with a silver electrode is easily reacted with hydrogen sulfide gas that microorganisms produce [37]. In this case, each measurement requires only a few minutes to detect LCFA with the QCM sensor. Therefore, the sensor can be operated in a batch mode to reduce exposure of the sensor to such a corrosive compound. From these reasons, it is believed that the QCM sensor is feasible for practical analysis of LCFAs in anaerobic digestate.