Food Waste Collection

Wearing disposable gloves, food waste was placed in a large self-sealing bag for mixing and crushing in the Weifang home kitchen. The mixed solid kitchen waste samples were randomly collected. Then, the sample was fully placed in 1.5 mL sterile centrifuge tubes, with masks and disposable gloves worn to avoid contamination of the samples during the sampling process.

DNA Extraction and Sample Delivery

Food waste samples were collected into 1.5 mL sterile centrifuge tubes and labeled on the outside of the tubes with time information. Laba (L); New Year Day (Yuandan, Y); Spring Festival Dinner (Nianye Fan, N); Daily (Richang, R); and All Celebrating Day (Festival, F), in which the daily meal was collected on 4 January 2023.

The solid kitchen waste samples were transported to the laboratory by being held in a 4ºC warm box and frozen using a -80ºC refrigerator. High-throughput microbial screening: According to the standard protocol, sample DNA was extracted using the FastDNA^TMSpin Kit for Soil (MP Biomedicals, Santa Ana, CA). PCR was performed using archaeal primers AR109F/AR915R and bacterial primers BA27F/BA907R, targeting the V4-V5 region of 16S rRNA [11]. The reaction parameters were as follows: pre-denaturation at 94ºC for 2 min; 94ºC denaturation for 30 s; annealing at 55ºC for 30 s. The reaction lasted for 25 cycles at 72ºC for 1 min. PCR products were detected via gel electrophoresis and then cut and purified for high-throughput sequencing.

The Quantitative Insights Into Microbial Ecology (QIIME) version 1.7.0 pipeline (http://www. Qiime. org) was used to process raw sequencing data with the default parameters [12]. Briefly, the representative sequences from each OTU were defined by a 97% identity threshold level, after which chimeric and low-quality reads were removed. Using the Ribosomal Database Project (RDP) classifier [13], the taxonomic classification of each OTU was assigned. The average relative abundance (%) of the predominant genus-level taxonomy in each sample was assessed by comparing the assigned sequence number of a particular taxon to the total obtained sequence number. To clarify microbial community differences, alpha diversity, PCoA, and Venn were computed with R [11].

The Bacterial Community in Food Waste

There were large differences between the microbial communities of household food waste at different festival times (Fig. 2), with L samples mainly dominated by Acinetobacter (50.66%), Pseudomonas (12.16%), Lactococcus (7.19%), Enterobacter (4.82%), and Vagococcus (2.16%), but Lactococcus especially was less common in household food waste samples from other festival times. Y samples were dominated by Acinetobacter (58.36%), Enterobacter (6.91%), Pseudomonas (6.26%), Weissella (2.50%), and Pantoea (1.96%). N samples were dominated by Pseudomonas (10.91%), Pectobacterium (1.35%), and Enterobacter (1.06%). R samples were dominated by Acinetobacter (20.71%), Pseudomonas (6.12%), with Aeromonas (1.39%) featuring the most. The results showed that the microorganisms were closer in the food waste on New Year Day and Laba, which may be presumed to be due to the closer proximity of Laba (30 December 2022) and New Year Day in 2023. The proximity in terms of meals was accompanied by the lack of complete waste sterilization after removal, so inoculation and perpetuation might happen. Chronologically, daily and Nian Ye meals were far away from both. At the same time, mitochondria and chloroplast DNA comprised a more significant portion of the diets on both daily (37.30%, 3.06%) and Nian Ye meals (79.10%, 25.40%). It was hypothesized that there might be an increase in the use of vegetables and fresh produce after Laba, resulting in higher levels of chloroplasts and mitochondria, representing green vegetation, in the food waste.

The high-throughput results have revealed large differences in the bacterial communities of food waste samples from Laba, New Year Day, Spring Festival, and Daily Meal. Among them, Pseudomonas was the most common among all samples, with a relative abundance ranging from 6.12%-12.16%, and widely distributed in various environments as a common gram-negative opportunistic pathogen [14]. The genus has good drug resistance and mutation ability and grows well in anaerobic and other extremely harsh environments. Acinetobacter, a common saprophytic microorganism, is a Gram-negative opportunistic pathogen in all food waste samples except for the N sample. This phenomenon may be because Spring Festival Dinners are mostly made of fresh food, fruits, and vegetables, increasing mitochondrial and chloroplast content while reducing the living space for spoilage microorganisms and decreasing their relative abundance. It can be seen that fresh ingredients not only effectively provide us with abundant nutrients [15], but also help to reduce opportunistic pathogens in food waste, and timely removal and disinfection of food waste can effectively protect us from the possibility of opportunistic pathogens [16].

Fig. 2.

Relative abundance of bacteria. Note: The raw data is plotted by taking the logarithm of lg. Laba (L), New Year Day (Yuandan, Y), Spring Festival dinner (Nianye Fan, N), Daily (Richang, R), celebrating days (Festival, F).

The Archaeal Community in Food Waste

The L sample was dominated by Methanosarcina (45.88%) and Methanobacterium (42.38%); the Y sample was dominated by Methanosarcina (15.07%) and Methanobacterium (5.87%); the R sample was dominated by Methanosarcina (96.40%) and Nitrososphaeraceae (3.31%); N was dominated by Methanobacterium (98.55%), of which, the relative abundance of Methanosarcina was higher in the L and R samples, but was below the limit of detection in the N samples (Fig. 3). Methanobacterium dominated the N samples and had a higher relative abundance (98.55%) in the N sample. The results showed that methanogens were the dominant genus in food waste samples at different times. Still, the composition of methanogens in food waste at different times was inconsistent.

Regarding archaea (Fig. 3), the methanogenic bacteria Methanosarcina and Methanobacterium, both of which are strictly anaerobic, dominated. Methanosarcina can complete methane production through three pathways: hydrogen, methyl, and acetic acid [14], but it requires a high concentration. In contrast, Methanobacterium is a formate-type methanogenic bacterium with specificity [17]. It can be surmised by the variation in the relative abundance of both in different samples that the abundance of starch on Laba Day provides the possibility for diverse organic matter. New Year’s Day is often not used as a major holiday. Therefore, the overall amount of litter may have been insufficient, leading to its low relative abundance of archaea. On the other hand, accumulating organic matter in daily life resulted in more diverse and abundant organic matter, providing better conditions for Methanosarcina to grow and multiply. However, preparing the annual dinner may have caused a certain randomness in the abundance increase of a single genus. Nitrososphaeraceae were present in all the samples but in low abundance, only having a relative abundance of 3.31% in the daily diet. As anaerobic archaea can be involved in the reduction of Fe(III) while oxidizing NH₄⁺ to produce nitrogen, nitrite, or nitrate [18], the greater variety and total amount of food waste generated from daily diets leads to the possibility of the existence of the above cycle in it, contributing to the degradation of food waste to form nitrogenous residues that can be used for restoration in the Gobi saline soil restoration [4].

The Analysis of the Bacterial Community in Food Waste

Principal Co-ordinates Analysis (PCoA) found (Fig. 4) that PCo1 and PCo2 explained 82.2% of the total sample variability, with 55.4% and 26.8%, respectively. In this case, the difference between bacteria in festive and daily food waste fell on the second principal axis, with an explanation of 26.8%. There were some differences between the bacterial communities in daily food waste and those at festivals, but the differences were greater among festivals due to different dietary needs.

Fig. 3.

Relative abundance of archaea. Note: The raw data is plotted by taking the logarithm of lg. Laba (L), New Year Day (Yuandan, Y), Spring Festival dinner (Nianye Fan, N), Daily (Richang, R), celebrating days (Festival, F).

Fig. 4.

PCoA analysis of bacterial community in food waste. Note: Daily (Richang, R), celebrating days (Festival, F).

Fig. 5.

Venn analysis of bacterial community in food waste. Note: Laba (L), New Year Day (Yuandan, Y), Spring Festival dinner (Nianye Fan, N), Daily (Richang, R).

From the overall view of the Venn diagram (Venn) analysis (Fig. 5), the total number of OTUs measured in the four samples was 6,439, and the total number of shared OTUs was 39. Among them, the highest number of OTUs in the L sample was 2,953; the lowest number of OTUs in the N sample was 671; the highest number of shared OTUs happened in the L and R samples was 441; and the lowest number of shared OTUs happened in the L and N samples was 52. The results showed that the bacterial communities in the food waste samples on the day of the Laba were more unique. At the same time, the dietary habits of Laba might be closer to those in daily routines, which resulted in more shared OTUs between samples that were further away from each other. Regarding bacterial colony diversity (Fig. 5), the bacterial communities of the festival and daily samples differed greatly, and the number of bacterial OTUs in the daily samples was lower than those in the samples of L and Y and higher than N.

Existing studies have found that the main purpose of food waste resources is to achieve organic energy and nutrient reuse while reducing environmental pollution and health and safety issues [3], consistent with the results of bacterial flora in this study. Pseudomonas and Acinetobacter are common opportunistic pathogens in food waste [14, 19], and the use of fresh ingredients can effectively provide more nutrients and reduce the proportion of pathogens in the flora, which can be reflected by the fact that Acinetobacter can degrade proteins rather than ferment sugars. The dietary structure of meat and eggs in China is more important. The composition of meat, eggs, and milk in our dietary structure is increasing significantly, providing conditions for opportunistic pathogenic bacteria [18]. Still, the timely removal of trash to ensure living safety has also become a noteworthy issue.

The Analysis of the Archaeal Community in Food Waste

PCoA (Fig. 6) analysis of the archaea revealed that the total explanation of inter-sample differences in both PCo1 and PCo2 axes was 85.9%, with the differences brought about by grouping being reflected in both PCo1 and PCo2 axes. This result suggests a significant difference in the archaeal community in food waste generated during holidays and usual diets, probably due to richer holiday diets.

Fig. 6.

PCoA analysis of archaeal community in food waste. Note: Daily (Richang, R), celebrating days (Festival, F).

Fig. 7.

Venn analysis of archaeal community in food waste. Note: Laba (L), New Year Day (Yuandan, Y), Spring Festival dinner (Nianye Fan, N), Daily (Richang, R).

Venn analysis of archaea revealed (Fig. 7) that the total number of OTUs measured in the four samples was 195, and there were no shared OTUs, but shared OTUs existed in the festival samples, which was only 1. The highest number of OTUs found in the Y samples was 79, and the lowest in the N samples was 43. The festival samples had the highest number of shared OTUs between L and Y at 19 and the lowest number of shared OTUs between L and N at 2. This result suggests that the number of OTUs in archaea is significantly smaller than that of bacteria and that temporal differences lead to greater variation in the archaeal community between samples.

The archaea are dominated by the methanogenic bacteria Methanosarcina and Methanobacterium, with Nitrososphaeraceae also prevalent. Thus, food waste can be cleaned with the help of methanogenic forms, and the waste can be resourced and rendered harmless by reducing its nitrogen content with the help of Nitrososphaeraceae [18]. In this progress, the nitrate-reducing bacteria coupled with anaerobic oxidation methanogens can use the methane produced by methanogens in kitchen waste, which realizes the simultaneous removal of nitrate and organic matter [17]. In this study, the open-ended collection of food waste in the bin did not facilitate the growth of anaerobic archaea. Still, rather than the growth of parthenogenetic opportunistic pathogens, a relatively anaerobic treatment environment would be more conducive to removing pathogenic microorganisms in future anaerobic food waste treatment to achieve harmless food waste recycling. However, the opportunistic pathogenic bacteria within food waste in real life are still a cause for concern, and the consumption of fresh vegetables, timely removal, and disinfection of waste are all key to ensuring human health [16].

Based on the results of the above studies, it was found that methanogens are important microorganisms that can be used for resource utilization in food waste from northern diets, but also as opportunistic pathogens Pseudomonas and Acinetobacter, which are parthenogenetic anaerobic microorganisms, but are less likely to be found than the methanogens Methanosarcina and Methanosarcina. Although both Pseudomonas and Acinetobacter are parthenogenetic anaerobic microorganisms, their anaerobic survival ability is still insufficient compared to the methanogens Methanosarcina and Methanobacterium, as well as the Nitrososphaeraceae, so food waste resource utilization can be achieved through the direct anaerobic oxidation of the food waste. Meanwhile, the high temperature of the Gobi in the summer helps the anaerobic oxidation of food waste to produce methane [10], which can be stored for winter living or produced on a large scale for industrial production, and the remaining residue can be used for vegetation restoration in the spring of the following year [4]. In addition, both Methanosarcina and Methanobacterium can cooperate with other microorganisms for methane production through electron transfer, especially direct interspecies electron transfer [17, 20], and this process can be effectively facilitated with the help of conductors such as biogenic carbon, magnetite, and nanoconductors.