Results and discussion

VFA concentration effect on E. coli inactivation

Volatile fatty acids are produced when larger organic molecules are hydrolysed and anaerobically oxidised to carboxylic acids. Depending on the alkalinity of the solution, this will affect the pH value. With a decrease in pH, the fraction of acids present in the non-dissociated form will increase, which are reported to support bacteria inactivation. In order to study the relationship between non-dissociated VFAs and E.coli inactivation, batch experiments were conducted with store-bought VFAs (acetate, propionate and butyrate at 2,000, 3,000 and 4,000 mg/L) and spiked in to 15 g/L TS UDDT-FS samples. Acetate, propionate or butyrate VFA concentration of 4000 mg/1 achieved E. coli inactivation to below detectable levels after two days of incubation.

Increasing the VFA concentration has a direct effect on E. coli inactivation (Figure 3.1): for butyrate, the E. coli log inactivation increases from 3 to 6 by increasing the spiking concentration from 2,000 to 4,000 mg/L. A similar trend was observed for the acetic acid and propionic acid spiked bottles. Increasing concentration leads to increase in ND-VFA fraction which increases the inhibitory effect.

The VFA chain length apparently had slight impact on E. coli inactivation as indicated by the calculated log inactivation rates (log inactivation per ND-VFA (meq/g TS added)). After one day treatment, with acetic, propionic and butyric acids, the achieved log inactivation, was 2.15, 2.13 and 2.11, respectively, showing a slight decrease with increasing chain length of the fatty (Figure 3. 2). Related studies investigating the effect of VFA chain length on Salmonella typhimurium and Vibrio cholera inactivation reported that inactivation efficiency decreased with increasing chain length (Goepfert & Hicks, 1969b; Kunte et al., 2000; Salsali et ah, 2006), which agrees with the trend observed in our present study for E. coli inactivation. Acetic acid, due to its lower molecular weight, diffuses faster across the bacterial membrane compared to propionic and butyric acid under the same conditions of temperature and pH. Upon passage of ND-VFA’s through the cell membrane of microbes they dissociate internally thus disturbing internal pH, impacting protein’s tertiary structure, and inhibiting microbial growth (Jiang et al, 2013; Wang et al, 2014a; Zhang et al, 2005), thus inactivating pathogens. Under neutral pH conditions, increased VFA toxicity with increasing chain length on methanogenesis has been

described previously (Van Lier el cil., 1993). The increased toxicity was ascribed to the presence of a longer a-polar aliphatic tail that more easily interferes with the bacterial or archaeal membrane.

Experimental results applying a set VFA concentration showed that the E. coli inactivation rate increased in time during the incubations: higher E. coli inactivation was achieved on the second day of treatment (Figure 3.1). The E. coli log inactivation achieved at 3000 mg/L butyric acid, after day 1 and day 2 of treatment was 4.2 and 6.0, respectively. The increased inactivation rate coincided with a drop in pH from 4.8-4.3 between day 0 and day 2 and a corresponding increase iir ND-VFA concentration from 21.6-33.9 meq/1. The pH drop from 4.8-4.3 increased protonation of the organic acids thus higher ND-VFA fraction in the waste sample with the overall effect being increased inhibitory. In addition, increased incubation time leads to longer exposure time of E. coli to toxic effect of the ND-VFA.

The control sample with UDDT-FS also showed a higher log inactivation rate at lower pH: at 4.8, the E. coli log inactivation was 1.2 log/d, while at pH 6.2 the inactivation did not exceed 0.6 log/d. The total amount of ND-VFA increased up to 0.5 meq/ g TS added in UDDT- FS, pH) s and 0.2 meq/ g TS added in UDDT-FS, pHun- hi the ЛТА spiked incubations, a lower pH was achieved at higher initial VFA concentration.

pH and E. coli inactivation trends at varying initial spiked concentrations of acetate, propionate and butyrate

Figure 3.1: pH and E. coli inactivation trends at varying initial spiked concentrations of acetate, propionate and butyrate: A) Day 1 of treatment, B) Day 2 of treatment

Build up and effect of ND-VFA on E. coli inactivation at various concentrations of acetate, propionate and butyrate on; A) Day 1 and B) Day 2 of treatment О MW hydrolysis

Figure 3.2: Build up and effect of ND-VFA on E. coli inactivation at various concentrations of acetate, propionate and butyrate on; A) Day 1 and B) Day 2 of treatment О MW hydrolysis: The used OMW hydrolysed rapidly, leading to a pH of 3.3 after 24 hours incubation. From an average initial E. coli count of 1.75E+07 ±1.3E+07, values were already below detectable levels after the first day. At the measured pH, over 97% of the total VFA was present in non-dissociated form, leading to a ND-VFA concentration of 36.4 meq/1. For OMW, the presence of readily degradable fractions of organics caused a rapid acidification leading to a high concentration of volatile fatty acids and a low pH. The latter is of interest for co-digestion with dried faecal matter since it will accelerate pathogen inactivation, particularly in two-stage or plug flow digestion systems. Further experiments were conducted to assess the potential of OMW co-digestion for UDDT-FS digestion.

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