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SCUM License Key
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Your septic tank includes a T-shaped outlet which prevents sludge and scum from leaving the tank and traveling into the drainfield area. Your tank should be pumped if the bottom of the scum layer is within six inches of the bottom of the outlet, if the top of the sludge layer is within 12 inches of the outlet, or if more than 25% of the liquid depth is sludge and scum.
Scum is formed by the adsorption of long-chain fatty acids (LCFAs) onto biomass surface in anaerobic digestion of oily substrates. Since scum is a recalcitrant substrate to be digested, it is disposed via landfilling or incineration, which results in biomass washout and a decrease in methane yield. The microbes contributing to scum degradation are unclear. This study aimed to investigate the cardinal microorganisms in anaerobic scum digestion. We pre-incubated a sludge with scum to enrich scum-degrading microbes. Using this sludge, a 1.3-times higher methane conversion rate (73%) and a faster LCFA degradation compared with control sludge were attained. Then, we analyzed the cardinal scum-degrading microbes in this pre-incubated sludge by changing the initial scum-loading rates. Increased 16S rRNA copy numbers for the syntrophic fatty-acid degrader Syntrophomonas and hydrogenotrophic methanogens were observed in scum high-loaded samples. 16S rRNA amplicon sequencing indicated that Syntrophomonas was the most abundant genus in all the samples. The amino-acid degrader Aminobacterium and hydrolytic genera such as Defluviitoga and Sporanaerobacter became more dominant as the scum-loading rate increased. Moreover, phylogenic analysis on Syntrophomonas revealed that Syntrophomonas palmitatica, which is capable of degrading LCFAs, related species became more dominant as the scum-loading rate increased. These results indicate that a variety of microorganisms that degrade LCFAs, proteins, and sugars are involved in effective scum degradation.
Citation: Sakurai R, Takizawa S, Fukuda Y, Tada C (2021) Exploration of microbial communities contributing to effective methane production from scum under anaerobic digestion. PLoS ONE 16(9): e0257651.
To reuse the collected scum as energy resources, several studies have employed scum as a co-digestion substrate with sewage sludge [7], thickened activated sludge, and primary sludge [3, 8]. The development of efficient scum digestion methods enables reduction of the cost of disposal and increase in biomethane yield. If anaerobic scum digestion can be promoted, the application range of anaerobic digestion can be expanded to include more oily substrates. However, the key microbes that play an important role in anaerobic scum digestion are unclear.
Our microbiological findings on anaerobic scum digestion will enable new approaches to solve the problems caused by scum. In this study, we aimed to reveal the microbes contributing to effective scum degradation. We pre-incubated a collected sludge with scum to enrich scum-degrading microbes. Additionally, to investigate the core scum-degrading microbes in this pre-incubated sludge, we evaluated the changes in microbial communities according to various loading rates.
Seed sludge was collected from a mesophilic food waste treatment plant at Tohoku University (Osaki, Japan). Before incubation with scum, this sludge was incubated with olive oil (1 mL L-1) at 35C in a 2-L screwcap bottle (1,800 L working volume). The consumption of the substrate was confirmed by the cessation of biogas generation [11]. When biogas production ceased, 2/3 of the culture was exchanged with fresh medium and seed sludge (1:1 v/v). The medium consisted of 0.14 g L-1 of KH2PO4, 0.54 g L-1 of NH4Cl, 0.20 g L-1 of MgCl26H2O, 0.15 g L-1 of CaCl22H2O, 2.5 g L-1 of NaHCO3, and 0.20 g L-1 of yeast extract (Difco, Detroit, MI, USA). Trace element and vitamin solutions were also used; details are provided in NBRC No. 398 (NITE Biological Resource Center, Chiba, Japan). After approximately 2 years of incubation, the olive oil was replaced with scum, which was collected from the oil separation tank before treatment of the food wastewater. The chemical oxygen demand (COD) of the scum was 280 g kg-1. Granular activated carbon was then added to promote methane production [12], and the sludge was incubated at 35C for approximately 6 months (Sludge I). Another sludge was collected from a mesophilic biogas plant at Tohoku University (Osaki, Japan) and incubated with 2 g L-1 glucose at 35C until use to avoid starvation. After cessation of biogas production was observed, this sludge was used in Experiment 1 (Sludge II).
The scum used for the batch experiments was collected from a mesophilic food waste treatment plant (Tokyo, Japan) and stored at 4C. The characteristics of scum and sludge are shown in Table 1. A total of 40 mL of Sludge I or Sludge II, 3.0 g of granular activated carbon, and 40 mL of NBRC No. 398 medium mentioned above were placed into a 100-mL vial. Scum was then added to adjust the concentration of 8.6 g COD L-1. The vials were purged with nitrogen gas to remove oxygen. The batch experiments were conducted in sextuplicate until day 8, and three of these replicates were opened on day 8 for DNA extraction and LCFA analysis. Then the batch experiments were continued using the remaining triplicates.
Batch experiments were conducted in parallel under the same conditions as those employed in Experiment 1. A total of 40 mL of Sludge I, 3.0 g of granular activated carbon, and 40 mL of NBRC No. 398 medium mentioned above were placed into a 100-mL vial. The initial scum loading concentrations were as follows: 8.6 g COD L-1, 12.6 g COD L-1, and 17.3 g COD L-1. The batch experiments were conducted in sextuplicate until day 8, and three of these replicates were opened on day 8 for DNA extraction and LCFA analysis. Then, the batch experiments were continued using the remaining triplicates. A schematic representation of this study is given in Fig 1.
The cumulative methane production of each sludge is shown in Fig 2A. Until day 7, the methane production of Sludge I was 30 1 mL g-1 VS. Then it increased to 121 1 mL g-1 VS on day 11. In contrast, the methane production of Sludge II was only 19 3 mL g-1 VS until day 11. The cumulative methane production on day 11 of Sludge I was 6-fold higher than that of Sludge II. Finally, 582 27 and 457 5 mL g-1 VS methane were produced from Sludge I and Sludge II, respectively. Methane conversion rates of Sludge I and Sludge II calculated from COD were 73% 3% and 57% 1%, respectively. Sludge I showed relatively high methane productivity from scum. The methane gasification process of scum was fitted with the modified Gompertz model in all samples (RMSE = 21 6). The theoretical methane production of Sludge I and Sludge II calculated by the modified Gompertz model was 630 25 and 470 6 mL g-1 VS, respectively. Fig 2B shows the transition of total VFA concentrations. On day 0, total VFA concentrations were 60 6 and 87 4 mg L-1 in Sludge I and Sludge II, respectively. In Sludge I, VFA concentration then decreased gradually and became undetectable on day 22. In Sludge II, the VFA concentration gradually increased to 216 13 mg L-1 on day 15. Among them, acetate was the dominant VFA (202 13 mg L-1). The concentration then decreased gradually and became undetectable on day 30. Considering the low methane production in Sludge II, it was inferred that acetate conversion to methane was inhibited until day 15. Methanogens are more susceptible to LCFA toxicity than acetogens [29]. In particular, acetoclastic methanogens have been reported to be more sensitive to LCFAs than hydrogenotrophic methanogens [4, 30]. It was inferred that LCFAs degradation in Sludge I was faster than that in Sludge II.
Our results confirmed that Sludge I had a relatively high potential for methane gasification of scum. It was assumed that scum-degrading microorganisms were enriched in Sludge I. To reveal the core scum-digesting community, the differences in microbial communities were evaluated according to the scum loading concentration.
Cumulative methane production is shown in Fig 4A. During the 30-day incubation, 582 27, 607 8, and 563 22 mL g-1 VS methane were produced from 8.6, 12.6, and 17.3 g COD L-1 scum-loaded vials, respectively. The methane gasification process of scum was well fitted with the modified Gompertz model in all samples (RMSE = 41 13). The theoretical methane productions calculated by the modified Gompertz model were 643 37, 667 7.3, and 630 25 in 8.6, 12.6, and 17.3 g COD L-1 scum-loaded vials, respectively. The methane conversion rates in 8.6, 12.6, and 17.3 g COD L-1 loaded vials were 73% 3%, 77% 1%, and 70% 3%, respectively. There were no significant differences between these values (TukeyHSD, p >0.05). The total VFAs gradually decreased in all the samples (Fig 4B). Temporal VFA accumulation was not observed. The total LCFA concentrations in 8.6, 12.6, and 17.3 g COD L-1 loaded vials were 1,142 179, 4,987 222, and 6,869 585 mg L-1, respectively (S2 Fig). Finally, most LCFAs were degraded in all the samples. Total LCFAs on day 30 in 8.6, 12.6, and 17.3 g COD L-1 loaded vials were 3.3 0.3, 5.7 0.3, and 19.3 8.0 mg L-1, respectively. Remarkably, the total LCFA concentrations in 12.6 and 17.3 g COD L-1 loaded vials were much higher than that in Sludge II (Experiment I), in which VFA accumulation and lower methane productivity was observed. Even though high concentrations of LCFAs were detected in scum high-loaded vials, inhibitory effects on scum degradation and methane productivity were not observed in comparison with 8.6 g COD L-1 loaded vials. VS decomposition rates were 73% 1%, 74% 1%, and 71% 2% in 8.6, 12.6, and 17.3 g COD L-1 loaded vials, respectively (S3A Fig) (TukeyHSD, p >0.05). Dissolved COD in each vial gradually decreased. Finally, the dissolved COD concentrations in 8.6, 12.6, and 17.3 g COD L-1 loaded vials were 68.3 5.9, 94.7 2.0, and 95.3 8.0 mg L-1, respectively (S3B Fig) (TukeyHSD, p >0.05). These results suggested that the scum digestion process was not inhibited in high-loaded samples. The inhibition caused by LCFAs has been reported as a reversible process [29]. In this study, LCFA-oxidizing bacteria may have degraded the LCFAs adsorbed onto methanogens smoothly. Methanogens and Syntrophomonas were quantified to assess the effect of various loading rates on these microbes. 2ff7e9595c
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