Co-digestion of hybrid Pennisetum and peanut shell after adding TiO 2 nanoparticles: Focusing on the synergistic effects on methane production

Anaerobic digestion is a widely accepted method to treat wastes such as peanut shell. The energy and nutrients are simultaneously recovered by this method. The objective of this study was to elucidate the effect of TiO 2 nanoparticles in co-digestion of hybrid Pennisetum and peanut shell under mesophilic conditions. The results demonstrated the met hane (CH 4 ) production was improved by adding the TiO 2 nanoparticles. The cumulative ga s production is best (up to 11,133.3 mL) by adding 0.15% nano-TiO 2 particles. The micro bial community analysis showed that Methanobacterium and Methanosarcina were enriched in the presence of TiO 2 nanoparticles indicating that TiO 2 can improve CH 4 production by stimulating the growth of methanogens.


Introduction
The total annual output of peanuts in China is 5 million tons. Peanut shells (PS) are food waste, and food waste constitutes 30-50% of municipal solid waste [1][2] . Approximately 60 million tons of food waste was produced last year in China alone. Food waste production is expected to keep increasing while maintaining social/economic growth as well as population growth.
Hybrid Pennisetum (HP) is herbaceous plant with great energetic potential. It has a high biological yield, strong regeneration ability, and multiple rounds of harvest [3][4] . Many researchers have focused on methane production from anaerobic fermentation of hybrid Pennisetum. However, Pennisetum is not fully utilized when used as a carbon source. Therefore, significant effort is needed to handle ever-increasing peanut shell waste and use it to produce biogas via Pennisetum.
Aerobic digestion has been developed and is well recognized as a promising tool for waste stabilization and energy recovery in the form of biogas. It has two processes: wet anaerobic digestion and dry anaerobic digestion. The total solid of wet anaerobic digestion system is less than 15%, and the total solid of dry anaerobic digestion system is higher than 15% [5][6] . Dry anaerobic digestion (DAD) is increasingly popular. This requires less water, and it does not cause more pollution than a wet digestion system [7][8][9] . In addition, co-digestion can greatly improve specific methane yields and methane production rates versus mono-digestion because of the superior nutrient availability and synergistic microbiomes.
Nanoparticles offer unique physiochemical properties and widespread applications [10] . Their effects on the environment have been investigated, but most studies focused on soil and wastewater toxicity [11][12] . The effect of nanomaterials on wastewater treatment has been reported [13][14][15] . The adsorption of activated sludge was reported to be the main mechanism of nanoparticles.
Nevertheless, the effects of metal oxide nanoparticles (such as TiO2 nanoparticles) on anaerobic digestion for HP and PS are rarely investigated. TiO2 nanoparticles have no significant toxicity on the viability of bacterial cells and show no inhibitory effects on waste-activated sludge digestion.
In this study, dry anaerobic experiments were conducted under mesophilic conditions with different mixing ratios of HP and PS. The purpose was to investigate methane production with different ratios of HP and PS, investigate the effects of five dosages of the TiO2 nanoparticles on methane yield, and assess the influences of TiO2 nanoparticles on flora.

Feedstock and inoculum
The surface part of hybrid Pennisetum used here was from the experimental farm of Yunnan Agricultural University, Kunming, Yunnan Province. The samples were cut into 2-3 cm. The dehydrated sludge was taken from the fifth water purification plant in Kunming, Yunnan Province, and appeared brown. The total solids (TS) content was 15.46%, and the volatile solid (VS) content was 41.54%.

Nanoparticles synthesis
Nano-TiO2 powder was synthesized according to a reported route [16] . Here, 0.1600 g (0.84 mmol) of dopamine (3,4-dihydroxy-β-phenylethylamine) and 30 mL of benzyl alcohol were added to an Erlenmeyer flask and stirred vigorously for 20 min. We then added 1.5 mL of TiCl4 dropwise. This was stirred vigorously at room temperature for 2 h. The temperature was then increased to 80°C, and the mixture was transferred to a polytetrafluoroethylene reactor at 80°C for 3 days. After cooling, the resulting red-brown mixture was centrifuged, the supernatant was decanted, and the precipitate was washed three times with chloroform and dried at 60°C to obtain a large amount of dark red nano-TiO2 powder.

DNA extraction and PCR amplification
DNA extraction of different digestion period samples was detected by a MIO-BIO Power Soil DNA Isolation Kit. The remaining steps for DNA extraction were performed via the DNA isolation kit protocol. Subsequently, the V4-V5 variable region of the bacterial 16S rRNA gene was amplified using primers 515F and 926R through polymerase chain reactions (PCRs). PCR was performed in a 10 μL volume containing 1× PCR buffer, 1 μL dNTPs, 1 μL primer, 1 unit taq DNA polymerase, and 5-50 ng template DNA under the two cycling conditions: Bacteria had pre-denaturation at 94°C for 2 min, 22 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s or 50°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 5 min. Archaea had pre-denaturation at 94°C for 2 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 5 min. Finally, the PCR products were tested through high-throughput sequencing using the Illumina Miseq 2x300bp platform.

Experimental set-up
The digestion device consists of a 1 L reaction bottle, gas bottle, water bottle, and constant-temperature water bath. According to the experimental design, different ratios of substrates and TiO2 nanoparticles were added to the reactors. All rectors were incubated at 37(±1°C).

Feedstock
The carbon to nitrogen ratio (C/N) plays an important role in dry AD. If the C/N is higher, then the gas yield may be lower. The methanogenic bacteria will be toxic if the C/N is lower. According to Weiland and Richa, a C/N ratio in the range of 20%-30% is the best. The C/N ratio of hybrid Pennisetum is 31.15 (C 47.35% and N 1.52%). The C/N of peanut shell was also measured ( Table   1). The C/N ratio is 25. We choose the hybrid Pennisetum and peanut shell as feedstocks.

Experimental design
The digestion process contains two steps. First, to determine the best ratio, the HP:PS mixing ratios were selected as 0:4, 1:3, 2:2, 3:1, and 4:0. Each sample group was set up in triplicates. Second, different quality scores of TiO2 nanoparticles were added to the reactors under the best ratio of HP:PS.

Analytical methods and characterization
The volatile solids (VS) and the total solids (TS) were measured by standard methods [17][18] . Total carbon (TC) and total nitrogen (TN) were measured with an elemental analysis instrument (VARIOEL Ш, Germany). The 16s rRNA gene amplification and sequencing used NGS Illumina MiSeq 2 x 300 bp. Fig. 1 shows 40-day methane yields for different ratios of mixed feedstock and cumulative methane production. The pH values in reactors ranged from 6.5 to 7.5 because they were suitable for

Sample
The content of elements

Impact of nano-TiO2 on methane production
The effects of TiO2 nanoparticles on anaerobic digestion are shown in Fig. 2a. The pH values in the reactors also ranged from 6.5 to 7.5. Five sets of experiments with different amounts of TiO2 starting from 0 to 2% were performed. The results showed that the addition of TiO2 had an encouraging impact on methane production. Under TiO2 nanoparticles addition, the cumulative methane production increased significantly. When 0.15% TiO2 was added, the cumulative methane production increased by 23.7%. However, the methane production decreased when the concentration of TiO2 was 0.20%. This indicated that the high concentration of TiO2 NPs inhibits anaerobic digestion. The COD curves of different samples on anaerobic digestion are shown in Fig.   2b. The trend of COD decreased after 10 days. The nitrogen and carbon content is exhausted along with the aging of the microbial cells. Moreover, the COD removal efficiency was gradually increased, and the order of COD removal rates was blank (57%) < 0.05% TiO2 (60%) < 0.1% TiO2 (62%) < 0.15% TiO2 (65%) < 0.2% TiO2 (67%).
(a) (b) Fig. 2 The curves of cumulative methane production (a) and COD (b).

Community diversity of archaea
Archaeal community diversity of three samples with different fermentation periods is shown in Table 2
Therefore, the categories of major archaea genera were similar, but their relative abundances were different. The archaea of Methanobacterium and Metgabisarcina were enriched with the addition of TiO2 nanoparticles. These results indicate that TiO2 played an important and positive role in CH4 production.

Conclusion
This study indicated that the addition of TiO2 nanoparticles promoted methane production in the system of anaerobic co-digestion of hybrid Pennisetum and peanut shell. In our future work, the effect of nanoparticles on fermentation will be further investigated, and biological flora will be studied in the fermentation process.