The pungency impact of 6-gingerol on the methane generation from food waste by anaerobic digestion: batch and two stages performance

doi:10.21203/rs.3.rs-1992137/v1

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The pungency impact of 6-gingerol on the methane generation from food waste by anaerobic digestion: batch and two stages performance

https://doi.org/10.21203/rs.3.rs-1992137/v1

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6-gingerol, a spicy active ingredient present in ginger, widely exists in food waste. Herein, the mechanism followed for the anaerobic digestion of 6-gingerol in food waste has been investigated. Experimental results showed that 6-gingerol could enhance methane production from 0.80% to 18.32% under a concentration of 10-30 mg/gVS. However, the cumulative methane yield decreased by 66.28% and 71.86% when the 6-gingerol concentration was 40 and 50 mg/gVS, respectively. The concentrations of volatile fatty acids, soluble chemical oxygen demand, carbohydrates and proteins in 6-gingerol-treated groups were slightly higher than in control, indicating that 6-gingerol might facilitate the release of organics into mixed liquor. Sequencing analysis revealed that the addition of 6-gingerol could inhibit the growth of acetoclastic methanogens and enhance the abundance of hydrogenotrophic methanogens. Studies on different stages showed that the extent of inhibition of methanogens associated with 6-gingerol was higher than that of the acidification stage.

Anaerobic digestion

Food waste

Methane generation

6-gingerol

Microbial communities

Food waste (FW) is the main component of organic solid waste, accounting for up to 50–60% of China’s municipal solid waste (Shah et al., 2021; Wang et al., 2016). Inappropriate disposal of FW can cause serious environmental problems, such as soil and groundwater pollution (Luo et al., 2019). Currently, composting, landfilling and other methods are widely used for the treatment of FW (Chen et al., 2020). However, serious environmental issues, high processing costs and huge land demands have severely restricted its application. Methods to address the problem of increasing the quantity of FW should be urgently developed. As a result, more and more attention is being paid to the treatment and recycling of FW (Chen et al., 2020). Among the various processing technologies, anaerobic digestion can not only support high organic burden but also obtain clean energy-biogas (Yu et al., 2018).

Numerous inhibitory factors are involved for the FW treatment as the response process associated with anaerobic digestion is complex such as C/N ratio, salinity, heavy metals, etc. (Li et al., 2022; Zheng et al., 2021). In addition, byproducts and intermediates produced during anaerobic digestion, such as free ammonia, volatile fatty acids (VFAs) and sulfides, which can also act as inhibitors (Jin et al., 2021; Zhang et al., 2020). Meanwhile, researchers have conducted the studies on new inhibitors such as microplastics (Azizi et al., 2021).

Generally, FW is primarily composed of carbohydrates, proteins and fats, making it readily biodegradable (Suarez et al., 2022). In addition, some spices are usually added when cooking in order to make food delicious, such as pepper, onion, garlic, ginger, etc. However, a small part of these spices are eaten as food, most of these are disposed as FW. Du et al. (2021) reported that the capsaicin content in capsicums usually ranges from 0.1 to 2.5 mg/g indicating that capsaicin accumulation in FW could be up to 25 g/kg. Global ginger production was about 3.3 million tons, there would be a lot of ginger waste to be disposed during processing and consumption (Gao et al., 2021). A large amount of ginger residues from the ginger byproduct processing plant are sent to the waste treatment station as solid waste for disposal. To date, researchers have studied the inhibition of allicin and capsaicin in the anaerobic digestion of organic solid wastes, while the interaction between gingerols and anaerobic digestion has been rarely indicated. Ginger, including organic acids, fibers and lipids, is a plant rich in several functional compounds, but the phenolic compounds as gingerols and shogaols are the major contributors to its bioactivity (23–25% and 18–25%, respectively) (Dalsasso et al., 2022; Sampath et al., 2021). Promdam et al. (2022) found that ginger rhizomes purchased from Thailand contain 17.0 mg/g of 6-gingerol standardized by HPLC. Roots contain 6-gingerol, the most abundant phenolic compound, which exhibits anticancer activity against a variety of cancers, such as colon, breast, skin and myeloid leukemia (Chatupheeraphat et al., 2020). High concentrations of 6-gingerol have found to be toxic due to its chemical structure (Lima et al., 2019). The 6-gingerol analogs function as potent biofilm inhibitors by inhibiting the formation of low-concentration Pseudomonas aeruginosa biofilm through quorum sensing disrupting activities (Ham et al., 2019). Further studies have found that the antibacterial effects of capsaicin and gingerols are reflected by their ability to inhibit the R-plasmid conjugation processes (Oyedemi et al., 2019).

So far, research on 6-gingerol was limited to the field of medical and dietary. The effect of this compound on the batch and two different stages of anaerobic digestion remained to be studied. This paper aimed to evaluate the effect of 6-gingerol on the yield of methane generation, the process of organic matter release, key enzyme activity and microbial community succession. Furthermore, the effect of the 6-gingerol dosage on methane generation was investigated. Finally, the effect of 6-gingerol on two different stages performance from FW by anaerobic digestion was explored. The results reported herein help in understanding the impact of 6-gingerol and its degradation on the process of anaerobic digestion of FW.

2.1. Source and characteristic of experimental materials

Inoculum sludge applied in this work was obtained from an anaerobic digester of a food factory located in Wuxi, China. The inoculated sludge was cultured in Erlenmeyer flasks at 37°C for 5 days prior to use. Each ingredient of FW (cabbage, 350 g/kg; rice, 400 g/kg; tofu, 50 g/kg; pork, 200 g/kg) was obtained from a supermarket in Wuxi. Each material was mixed in the above ratio after it was cooked (Tao et al., 2021). The detailed properties of the substrate and inoculum were shown in Table S1. The 6-gingerol was purchased from Chengdu Biopurify Phytochemicals Ltd with a purity of 96%. The 6-gingerol had a molecular weight of 294.39 g/mol, a molecular formula of C₁₇H₂₆O₄, and was stored in the dark at -20°C prior to experiments.

2.2. Effect of 6-gingerol on batch and two-stages methanogenesis

2.2.1. Effect of 6-gingerol on the batch experiment

The batch test was performed in 500 mL serum bottles under mesophilic conditions (37 ± 0.5 ℃). The initial pH value of the reaction was adjusted to 8.0 ± 0.2 by using 3 mol/L sodium hydroxide and 3 mol/L hydrochloric acid solutions (Yang et al., 2015). The ratio of food waste to inoculation sludge in this paper was 1:1.5 (based on VS) (Fagbohungbe et al., 2015). Different concentrations of 6-gingerol (0, 10, 20, 30, 40 and 50 mg) were added to 1 gVS of FW with a final FW of 8 gVS, which were marked as K1, A1, A2, A3, A4 and A5, respectively (Promdam et al., 2022). In this paper, concentrations of 10–30 mg/gVS (group A1-A3) were defined as low concentration and concentrations of 40–50 mg/gVS (group A4 and A5) were defined as high concentration. Each bottle was flushed with high purity nitrogen gas for 5 minutes to ensure an anaerobic reaction environment.

The automatic methane potential test system (AMPTS II) was used to analyze methane production (Li et al., 2020). Every group was conducted in triplicate.

2.2.2. Effect of 6-gingerol on the two different stages

The methane production process from FW with 6-gingerol addition was primarily proceeded over acidification of FW and methanation stages (Mu et al., 2012). The tests were performed to study the effect of 6-gingerol concentrations (10, 20, 30, 40 and 50 mg/g VS) on the two different stages.

Experimental setup for the acidification process

The influences of 6-gingerol addition on the acidification stage were assessed following the procedure reported in Section 2.2.1. Before the reaction, the anaerobic sludge was antoclaved for 30 min at 105℃ to ensure that only acid-producing bacteria are present. The anaerobic inoculum (12 gVS sludge and 8 gVS of the substrate; 400 mL) was added into each of serum bottles for testing. After purging with high purity nitrogen gas for 5 min, all bottles were sealed at 37 ± 1 ℃. The impact of 6-gingerol (10, 20, 30, 40 and 50 mg/gVS) on the acidification process was assessed by analyzing the efficiency of total volatile fatty acids (TVFAs). The TVFAs contents were monitored every 48 h.

Experimental setup for the methanation process

The effect of 6-gingerol addition on the process of methanogenesis was assessed by using sodium acetate (5 g/L) as the substrate (Dong et al., 2019) because the acetate-trophic methanogenesis process accounted for the majority of the overall methanogenesis process. This test was conducted following the processes described in Section 2.2.1. The difference lay in the fact that 5 g/L of sodium acetate was used instead of the substrate.

2.3. Analytical methods

The contents of volatile solid (VS), total solid (TS), soluble chemical oxygen demand (_SCOD) and ammonium-nitrogen were determined according to standard methods (APHA, 1995). The pH variation was monitored using a pH meter (ST3100M, USA). The content of carbohydrates was measured by the phenol-sulfuric acid method (Dubois et al., 1965). The content of crude protein was determined by the Kjeldahl method (Hall & Schonfeldt, 2013). The contents of C, N, H and S were measured based on the dry weight using a Flash 2000 elemental analyzer. The concentration of VFAs was determined using a gas chromatograph (GC-2010 plus, Shimadzu, Japan). The acetate kinase (ACK), dehydrogenase (DHA) and alkaline protease (AKP) enzyme activity kits were purchased from Suzhou Comin Biotechnology (China). The concentration of the coenzyme F₄₂₀ was determined using a UV spectrophotometer (Qi, 1997). The concentration of 6-gingerol was determined according to the high performance liquid chromatography (HPLC) analysis procedure reported by Np et al. (2022). Tightly bound extracellular polymeric substances (TB-EPS) and loosely bound extracellular polymeric substances (LB-EPS)were analyzed according to the extraction method reported by Zhang et al. (2016).

2.4. Analysis of the microbial community

At the end of the reaction, the samples for high-throughput sequencing analysis were collected and conserved at ‒80 ℃ for microbial community analysis in each group. Microbial deoxyribonucleic acid (DNA) was extracted using a PowerSoil kit (Sangon Biotech, Shanghai, China) in parallel samples. The polymerase chain reaction (PCR) technique was used to amplify the V3-V4 region of the 16S rDNA gene in the bacterial and archaeal community. Then, the DNA samples were sequenced using the Illumina Hiseq 2000 pyrosequencing platform. The universal primers of 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTA

AT-3′) were used to target bacteria. The 16S rDNA primers MLfF (5′-GGTGGTGTMG GATTCACACARTAYGCWACAGC-3′) and MLrR (5′-TTCATT

GCRTAGTTWG GRTAGTT-3′) were used to target archaea.

2.5. Statistical analysis

The significance of the results was assessed using analysis of variance (ANOVA) with the least significant difference test, and P < 0.05 was considered statistically significant whereas P ≥ 0.05 was considered to be nonsignificant.

3.1. Effects of 6-gingerol addition on the methane yield

The daily and cumulative methane yields under different concentrations of 6-gingerol were presented in Fig. 1. Since FW contained a lot of easily digestible components that could be degraded, the daily methane production ranged from 45.25 to 73.13 mL/g VS on the first day, indicating a rapid start-up of the reaction. Two methane generation peaks were observed in 37-days reaction. The rate of methane production rapidly increased in the first week, during which the daily methane yield peaked in all groups. This was attributed to the rapid biodegradation of the organic matters in FW in the early stage and then used for VFAs and methane production. The appearance of a second peak was observed, and the peak could be observed on the 14th day in the control. At the same time, it showed that the rate of methane production for groups A1-A3 reached a plateau on the 9th -24th days which was not observed in the control.

Meanwhile, for the treatment group with a concentration of 10 to 30 mg/gVS of 6-gingerol, the second stage of rapid increase associated with the cumulative methane yield was observed from the 15th to 30th day. The occurrence of the lag phase shown in this period could be potentially attributed to the addition of 6-gingerol. On the contrary, in the 40–50 mg/gVS dose groups (A4 and A5), the cumulative methane yield presented no substantial rise after the first week. This was probably because the pH of the system dropped rapidly after the organic matter in the FW was converted into VFAs, and the accumulation of which inhibited the process of biogas production. The rate of methane generation stabilized at the end of the experimental periods, indicating that a large amount of organic matter in the substrate had been utilized by microorganisms for methane production. At the end of the reaction, the cumulative methane yields corresponding to groups K1, A1, A2, A3, A4 and A5 were 488.63, 555.00, 578.13, 493.00, 164.76 and 137.50 mL/gVS, respectively. The results indicated that the addition of 6-gingerol into the anaerobic digestion system could decelerate the biodegradation of intermediates like VFAs, resulting in a lag in the gas production process. The microorganisms took time to adapt to the changes in the environment following the addition of 6-gingerol (Hall & Schoenfeldt, 2013). The results could be potentially attributed to the potential of excess 6-gingerol to suppress the function of the enzymes associated with methane production in microbes and stimulate the cells to secrete extracellular polymers. This helped reduce the membrane flux and the extent of transmission of substances inside and outside the cell.

3.2. Variation of pH and VFAs

The change in pH value and VFAs were presented in Fig. S1. The pH dropped drastically within the first two days. The lowest pH in each group was in the range of 7.31–7.59 on the 2nd day. This phenomenon could be explained by the initial degradation of organic matter in the substrate into VFAs by anaerobic microorganisms during the anaerobic digestion process. Subsequently, it gradually increased and reached a plateau attributed to the rapid utilization of intermediates such as VFAs by archaea. The reduction of acidity could cause the pH to rise. Subsequently, pH decreased for a second time and rose again until the 28th day. Finally, the pH remained stable until the end of the reaction. The final pH values were lowest in groups A4 and A5, and the values were less than 7.8. Meanwhile, there was no significant difference between the pH values corresponding to A1-A3 and the control groups whose values were all above 8.0.

During the first week of reaction, total VFAs initially increased and then dropped in all groups except A5. The total VFAs content in A5 continued to increase but the pH value picked up, which might be due to the buffering effect of sodium bicarbonate added before the reaction. The organic matter was efficiently degraded in all the groups, and this was reflected by an increase in the total VFAs levels around the 13th day (Fig. S1b). At this time, all groups except A5 reached the maximum total VFAs concentration after the start of the reaction. Groups K1, A1, A2, A3, A4 and A5 exhibited the peak values of 3.10, 1.50, 3.06, 5.59, 5.55 and 13.23 g/L on the 13th day, respectively. Much higher levels of VFAs have been reported for proteins and starches than other materials such as cellulose during the fermentation of putrefying waste (Strazzera et al., 2021). Therefore, the microorganisms mainly degrade the protein and starch in the substrates leading to the accumulation of acids, especially in A5, resulting in inhibited methanogenesis (see daily methane production profile in Section 3.1). The change trend of acetic acid was similar to that of TVFAs because acetic acid accounted for a large proportion of TVFAs. The concentration of butyric acid and propionic acid was low, the total content was less than 3 g/L and it did not change significantly during the whole reaction process. It was indicated that high doses of 6-gingerol significantly inhibited the VFAs degradation which resulted in the deceleration of the rate of their assimilation into methane. The accumulation of VFAs without a concurrent increase in the buffer capacity resulted in a decrease in the pH value that affected the anaerobic digestion process. The results indicated that the addition of 6-gingerol to the fermentation system could reduce the utilization of VFAs by methanogens. When 6-gingerol was added at a concentration of 40–50 mg/gVS, the final VFAs content was found to be higher than that in the control and low concentration groups. This could be attributed to the fact that the addition of 6-gingerol affected the efficiency of utilization of volatile fatty acids by methanogens. Under these conditions, the accumulation of total acids in A4 and A5 inhibited methanogenesis.

3.3. Analysis of sCOD, protein, carbohydrate, NH₄⁺-N and 6-gingerol

The concentration of sCOD with different doses of 6-gingerol was evaluated (Fig. 2a). The sCOD concentration increased rapidly during the first 2 days and reached the first peak on the 2nd day, while the maximum sCOD concentration in each group were 4060, 1248, 1856, 5728, 3648 and 5088 mg/L, respectively. From the 7th day to the end of the reaction, the sCOD concentration gradually decreased after reaching the second peak in the anaerobic digestion system. The intensity of the second peak was approximately 2 times higher than the first peak in K1 and A1-A3. On the contrary, the sCOD concentration continued to rise until the end of the reaction, and this was stimulated by concentrations of 40–50 mg/gVS. This could be attributed to acidification resulting in significantly high sCOD and low pH values. The two peaks could be attributed to the fact that FW was rich in proteins and starchy materials. Figure 2b presented that the soluble carbohydrate content continued to rise until half of the reaction. Then, the content slowly decreased till the final of the reaction. However, the carbohydrate content of the A3-A5 group decreased toward to the end. The content of the A4 and A5 group was much higher than that of the A1-A3 groups at the end of the reaction. The maximum carbohydrate contents in group A1-A5 were 114.32, 134.11, 241.12, 236.38 and 244.57 mg/L, respectively, which were 0.87, 1.02, 1.84, 1.80 and 1.87 times higher than the control, respectively. This could be attributed to that the high concentration of 6-gingerol inhibited the organic matter utilization and resulted in the carbohydrate accumulation. Previous study showed that anaerobic conversion protein to methane took a long time (Chiu et al., 2021). The protein accumulation reached the first peak around the 12th day. Then, the extent of accumulation gradually decreased until the second peak around the 30th day. Subsequently, it was used to produce methane until stability was reached. Compared with the control, the experimental group accumulated more proteins in the aqueous phase and the reason for this may be similar to that of sCOD. The result demonstrated that the solid protein in the FW was hydrolyzed into the liquid phase at the initial stage of the reaction, and the degradation of the microbial cell wall and the release of intracellular substances resulted in an increase in the concentration of soluble protein. The time required for the microorganisms to adapt to the environment after 6-gingerol addition resulted in a lag in the response.

The content of ammonia nitrogen gradually increased and stabilized on the 10th day (Fig. 2d). On the 22nd day, the concentration of ammonia nitrogen increased again until the end of the reaction. Finally, the ammonia nitrogen content for different groups was similar, with the values of 707.57, 722.48, 755.52, 779.12, 759.07 and 884.13 mg/L, respectively. The maximum content was in A5, and it could promote the hydrolysis of nitrogen-containing organic matter. Figure 2e presented the changes in the 6-gingerol content in the aqueous phase before and after the reaction. Before the reaction, the concentration of 6-gingerol in each group was 0, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/L, respectively. However, the substance could hardly be detected in the aqueous phase at the end of the reaction, and this could be attributed to the fact that 6-gingerol was degraded during the reaction.

3.4. Effect of 6-gingerol in LB-EPS and TB-EPS

EPS is a complex mixture of polymers in which polysaccharides and proteins account for the majority of the total organic matters (Zhou et al., 2020). As was shown in Fig. S2, the polysaccharides and proteins contents for LB-EPS in the control group were 30.94 and 82.99 mg/L, respectively. The LB-EPS in the low concentration experimental group was similar to that in the control. When the dosage of 6-gingerol was 40 and 50 mg/gVS, it could stimulate microorganisms to secrete more amounts of proteins. When the 6-gingerol content was 50 mg/gVS, the polysaccharides and proteins contents in LB-EPS were 37.84 and 201.55 mg/L, respectively. The content of polysaccharides and proteins increased with an increase in 6-gingerol. This revealed that 6-gingerol significantly promoted the process of secretion of proteins and organic matters.

The difference between LB-EPS and TB-EPS depends largely on the extraction method. Compared with LB-EPS, more severe conditions are required to extract TB-EPS from biomass. (Jahn & Nielsen, 1998). Compared to the content of polysaccharides, the proteins content was more dominant in TB-EPS production with 6-gingerol addition. The proteins content in TB-EPS was higher than polysaccharide with increasing 6-gingerol concentration, while the contents of polysaccharides showed a similar trend. In general, microorganisms released a mass of EPS for self-protection under unfavorable environments (Liu et al., 2016). Therefore, the increasing concentrations of polysaccharides and proteins in EPS indicated that the presence of 6-gingerol would cause certain stress to microorganisms. However, it has been found that the biofilms treated with 6-gingerol and its analogs had a 24–28% reduction in total carbohydrates and a 49–63% reduction in total proteins. Therefore, the reduction of EPS components by 6-gingerol and its analogs was beneficial to improve permeate flux in the reverse osmosis processes (Ham et al., 2019). Compared with the phenomenon reported above, the effect of 6-gingerol on polysaccharides and proteins in EPS in this paper was exactly different, this was probably because of the various anaerobic digestion system involved.

3.5. Microbial analysis in the reaction groups

3.5.1. Effect of 6-gingerol on the enzymes

The generation of methane is closely related to the activities of various enzymes associated with the process of anaerobic digestion. Each stage of anaerobic digestion performance involves the participation of key enzymes, such as AKP, ACK, DHA and coenzyme F₄₂₀ (Wang et al., 2022). Compared with the control, the activities of AKP were significantly increased with 6-gingerol exposure, especially in A1-A3 (Fig. S3). This phenomenon indicated that microorganisms stimulated by 6-gingerol secreted more proteins to the cell surface resulted in an increase of protein content in LB-EPS and TB-EPS. In detail, it was found that the addition of 6-gingerol positively correlated with the activities of ACK after the reaction. This was why the yield of acetic acid in the experimental group was higher than that in the control, especially in A4 and A5.

DHA is an extracellular enzyme that can reflect the change in microbial activity in the anaerobic digestion system. The change in its content is closely related to the process of oxidative phosphorylation in cells. Compared with the control, the effect of the groups A4-A5 on DHA was not significant (P༞0.05). It was observed that the low concentration of 6-gingerol promoted the activity of DHA, indicating that a small amount of 6-gingerol could promote cellular activity during methanogens resulted in increased methane production. In methanogenesis, H₂ and CO₂ can be converted into 5-methyl-THMPT, of which F₄₂₀ was the key enzyme. The increase of 6-gingerol promoted the activity of the coenzyme F₄₂₀, indicating that 6-gingerol could promote the process of hydrogenotrophic methanogenesis. Du et al. (2021) found that the presence of capsaicin inhibited the activity of F₄₂₀ and AK in acetotrophic and hydrogenotrophic methanogenesis. However, the effect of 6-gingerol on enzyme activity was different to that of capsaicin, which may be because 6-gingerol could promote enzyme activity and methane production at low concentration, while capsaicin at low concentration had a negative effect on anaerobic digestion system.

3.5.2. Microbial abundance at phylum level

Mixed anaerobic microbial communities significantly affect the valorization of waste biomass via the process of anaerobic digestion (Innard & Chong, 2022). As shown in Fig. S4, the seven dominant bacterial phyla were found to be similar in all tested groups. The main phylum bacterial strains of the control group were Firmicutes (24.08%), Bacteroidetes (23.25%), Chloroflexi (7.64%), Proteobacteria (12.91%), Synergistetes (2.07%) and Actinobacteria (3.57%). The relative abundances of Firmicutes in A1-A4 were 19.64%, 21.37%, 40.90% and 60.75%, respectively. It was observed that the abundance of Firmicutes dropped significantly to 28.88% when the concentration was the maximum (50 mg/g VS). This phenomenon suggested that when the concentration was in the range of 10–40 mg/gVS, 6-gingerol could stimulate the growth of Firmicutes, and when the concentration was 50 mg/gVS, an inhibitory effect was generated. Firmicutes could degrade macromolecules, such as lipids and proteins, resulting in the generation of acetic acid. Consequently, the enrichment of Firmicutes in A1-A4 could accelerate the degradation of complex organic matters during the hydrolysis stage.

As a kind of hydrolytic fermentation bacteria, Chloroflexi could accelerate the anaerobic digestion reaction (Zuo et al., 2020). Chiu et al. (2021) reported that the abundance of Chloroflexi could decrease with the increase in the concentration of organic components in the anaerobic digestion. The relative abundance of Chloroflexi was lowest in the control, and it accounted for only 7.64% of the total abundance. The relative abundance of Chloroflexi in A1-A5 was 7.26–13.47%. This was higher than the abundance recorded for the control group, indicating that 6-gingerol could effectively promote the abundance of Chloroflexi. Proteobacteria includes various cotrophic acetogenic bacteria that can convert propionic and butyric acid into acetic acid, CO₂ and H₂. The relative abundance of Proteobacteria was 12.91% in the control, and the relative abundance in the experimental groups was 1.96–2.35%. This was less than that reported for the control group. Actinobacteria were involved in the degradation of complex proteins and carbohydrates. They were also associated with acetic acid production during the fermentation stage. It has been found that Actinobacteria and Bacteroidetes could be growth promoters of acetoclastic archaea and syntrophic bacteria (Gulhane et al., 2017). The relative abundance of Actinobacteria in the control group was 3.57% while the abundance of Actinobacteria in the experimental groups was in the range of 0.46–3.04%. Zhang et al. (2021) reported that Actinobacteria was involved in the acidification process, resulting in the production of propionic acid. Therefore, the negative correlation of Actinobacteria with the concentration could be attributed to the fact that 6-gingerol can inhibit the propyl acidification process associated with FW.

In the hydrolysis and acidification stages, Bacteroidetes played an important role, which could convert sugar to acetic acid as the major product (Huang et al., 2019). The abundances of Bacteroidetes in A1-A5 were 21.31%, 13.8%, 15.59%, 8.75% and 9.03%, respectively. The abundance recorded for the control group was higher (23.25%) which indicated that 6-gingerol inhibited the growth of Bacteroidetes. Therefore, compared with the control group, the decrease in the abundance of Bacteroides in the experimental group also indicated that the polysaccharides in the FW were difficult to degrade, which lead to the accumulation of soluble polysaccharides in the digestive liquid and EPS at the end of the reaction. The total proportion of Bacteroidetes, Proteobacteria, Chloroflexi and Firmicutes associated with hydrolytic acidification changed significantly in the presence of 6-gingerol (P > 0.05) (Yavuztürk Gül et al., 2018).

3.5.3. Microbial abundance at genus level

The compositions and Beta diversity of archaeal methanogenic communities at the genus level were explored to better understand the effect of 6-gingerol on the microbial community structure (Fig. 3). The microbial community structures changed with the doses of 6-gingerol in the anaerobic digestion system. The archaea with clear classification information in the analytical database were Methanothrix, Methanobacterium, Methanolinea, Methanomassiliicoccus, Methanomethylovorans, Methanosarcina and Methanofollis. In general, Methanothrix played an important role in the anaerobic digestion system, and this could be attributed to their slow growth rate and high acetic acid affinity (Huang et al., 2019). It has been found that Methanothrix can not only metabolize acetic acid but also can accept electrons to reduce CO₂ to CH₄ (Zhu et al., 2020). Its relative abundance in the control group (58.97%) was higher than that in A2-A5 (38.46–57.40%), indicating that 6-gingerol had a stressing effect on the relative abundance of Methanothrix. Therefore, the decrease in the relative abundance of Methanothrix in the experimental group also indicated that the addition of 6-gingerol could inhibit the utilization of acetic acid, which was consistent with the accumulation of acetic acid in the experimental group (Fig. S1c). The second dominant genus was Methanobacterium (Euryarchaeota phylum) which belonged to the group of hydrogenotrophic methanogens (Hamana et al., 2009). The abundance of the hydrogenotrophic methanogens (Methanobacterium) in the control group was 29.77%, while the abundances in A1-A4 were 13.88–34.13%. However, the relative abundance of Methanobacterium was only 5.11% in A5. This could be potentially attributed to the fact that the methane generation pathway was more dependent on the conversion of H₂ and CO₂ to methane with the addition of 6-gingerol rather than acetotrophic methanogenesis.

The dominance of Methanothrix (38.46–68.26%) and Methanobacterium (5.11–34.13%) in all the groups demonstrated that hydrogenotrophic and acetotrophic methanogens played important roles during the conversion of organics into CH₄. It has been reported that Methanolinea was a mesophilic methanogenic bacterium capable of utilizing H₂/CO₂ (Sanae et al., 2012). Compared with the control group, hydrotropic methanogens in the experimental group gradually changed from Methanobacterium to Methanolinea when the 6-gingerol concentration increased. The addition of 6-gingerol stimulated the growth of Methanolinea, resulting in 6.23–24.95% abundance in the experimental group. This only accounted for 5.52% of the system in the control group. It was found that Methanomassiliicoccus is a hydrogenotrophic and methylotrophic archaea. The abundance of Methanomassiliicoccus was lower (< 1%) in both the control and low-concentration groups (i.e., in K1, A1 and A2). However, the relative abundance of Methanomassiliicoccus was significantly high in the high-concentration group (i.e., in A3, A4 and A5). The abundances were 6.51%, 1.68% and 3.50%, respectively. This phenomenon can be potentially attributed to the fact that the high concentration of 6-gingerol could promote the hydrolysis and acidification of organic matter, resulting in the release of a large amount of hydrogen that stimulates the growth of Methanomassiliicoccus. The relative abundance of Methanomethylovorans was dependent on the dosage of 6-gingerol, and the relative abundance of Methanomethylovorans increased with an increase in the dosage of 6-gingerol. Baselga (2017) and Lennon (2001) found that two components of species difference patterns can be described by analyzing beta diversity: (1) the disappearance or increase of species, known as species nesting component; (2) species replacement, also known as the species turnover component. With the increase of 6-gingerol concentration, the diversity gap between the experimental group and the control increased which was due to the fact that 6-gingerol could change the microbial environment and thus the diversity of microorganisms (Fig. 3b). This phenomenon also illustrated how 6-gingerol promoted and inhibited methane generation.

3.6. Effects of 6-gingerol addition on the acidification and methanation stages

Methane generation primarily proceeded over two stages: acidification of organic matter and methanization of organic acids. Hence, it was important to explore the effect of 6-gingerol on different stages involved in the process of methane generation. The effect of 6-gingerol on the acidification stage was presented in Fig. 4a. During the first 6 days of the reaction, the organic matter in FW was rapidly hydrolyzed and utilized to produce organic acids. Following this, the rate of acid production in each group increased slowly until the reaction was completed. At the end of the reaction, the TVFAs concentrations in each group were 12.67, 9.56, 12.55, 9.78, 10.21 and 10.76 g/L, respectively, indicating that the TVFAs concentrations in the experimental groups was not significantly different from the control (P ≥ 0.05). This indicated that the addition of 6-gingerol did not significantly affect the changes in bacteria (Fig. S4). The addition of 6-gingerol did not promote the acidification process, but had a more significant effect on the methanogenesis stage.

The results obtained from the methanization stage (Fig. 4b) were similar to the batch experiment (Fig. 1b). At the end of the reaction, the cumulative methane yield was 321.43, 211.27, 369.42, 165.49, 98.59 and 92.25 mL/g VS, respectively. Methane production in the low-concentration groups was significantly higher than that in the high-concentration groups. However, the difference in cumulative methane yield between the two stages and batch reactions of groups A1 and A3 might be caused by different substrates. In the high concentration group, the experimental group dosed with 40–50 mg/g VS of 6-gingerol produced less methane, which was similar to the batch experiment. The minimum amount of methane was produced at 50 mg/gVS of 6-gingerol which was only 28.10% of the control. The results revealed that an appropriate amount of 6-gingerol could promote the utilization of acid by archaea. It was observed that the process could be inhibited if the concentration was too high due to the decreased utilization of sodium acetate by archaea. Finally, it was demonstrated that the inhibition of FW on methanogenesis stage was significantly higher than that on acidogenesis stage. This could be potentially attributed to the fact that the addition of low concentration of 6-gingerol could increase the growth of acetic acid trophic methanogens, while a high concentration of 6-gingerol changed acidotrophic methanogen-dominant to hydrogenotrophic neo-methanogen-dominant.

The addition of 6-gingerol could help increase methane production during anaerobic digestion from food waste at a concentration of 10–30 mg/gVS. The 20 mg/gVS of 6-gingerol addition increased the maximum methane yield by 18.32% from food waste while the 50 mg/gVS addition inhibited by 71.86%. The polysaccharides and proteins in LB-EPS and TB-EPS increased gradually in 6-gingerol-treated groups. Futhermore, 6-gingerol could help improve the ACK and F₄₂₀ activities. The abundance of Chloroflexi and Methanobacterium increased following the presence of 6-gingerol. A certain concentration of 6-gingerol was beneficial for improving the hydrolysis and utilization of intermediates like VFAs.

Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Authors' contributions All authors participated in the planning, execution and finalization of the paper and approved the final version submitted.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (2018YFC1903000, 2019YFC1906304); Jiangsu Province Science and Technology Support Project of Social Development-General Program (BE2020755); the Fundamental Research Funds for the Central Universities (JUSRP22021).

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The pungency impact of 6-gingerol on the methane generation from food waste by anaerobic digestion: batch and two stages performance

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Source and characteristic of experimental materials

2.2. Effect of 6-gingerol on batch and two-stages methanogenesis

2.2.1. Effect of 6-gingerol on the batch experiment

2.2.2. Effect of 6-gingerol on the two different stages

2.3. Analytical methods

2.4. Analysis of the microbial community

2.5. Statistical analysis

3. Results And Discussion

3.1. Effects of 6-gingerol addition on the methane yield

3.2. Variation of pH and VFAs

3.3. Analysis of sCOD, protein, carbohydrate, NH₄⁺-N and 6-gingerol

3.4. Effect of 6-gingerol in LB-EPS and TB-EPS

3.5. Microbial analysis in the reaction groups

3.5.1. Effect of 6-gingerol on the enzymes

3.5.2. Microbial abundance at phylum level

3.5.3. Microbial abundance at genus level

3.6. Effects of 6-gingerol addition on the acidification and methanation stages

4. Conclusion

Declarations

References

Supplementary Files

Status:

Version 1

The pungency impact of 6-gingerol on the methane generation from food waste by anaerobic digestion: batch and two stages performance

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Source and characteristic of experimental materials

2.2. Effect of 6-gingerol on batch and two-stages methanogenesis

2.2.1. Effect of 6-gingerol on the batch experiment

2.2.2. Effect of 6-gingerol on the two different stages

2.3. Analytical methods

2.4. Analysis of the microbial community

2.5. Statistical analysis

3. Results And Discussion

3.1. Effects of 6-gingerol addition on the methane yield

3.2. Variation of pH and VFAs

3.3. Analysis of sCOD, protein, carbohydrate, NH4+-N and 6-gingerol

3.4. Effect of 6-gingerol in LB-EPS and TB-EPS

3.5. Microbial analysis in the reaction groups

3.5.1. Effect of 6-gingerol on the enzymes

3.5.2. Microbial abundance at phylum level

3.5.3. Microbial abundance at genus level

3.6. Effects of 6-gingerol addition on the acidification and methanation stages

4. Conclusion

Declarations

References

Supplementary Files

Status:

Version 1

3.3. Analysis of sCOD, protein, carbohydrate, NH₄⁺-N and 6-gingerol