Thermophilic Composting of Human Feces: Development of Bacterial Community Composition and Antimicrobial Resistance Gene Pool

Abstract

In times of climate change, practicing sustainable, climate-resilient, and productive agriculture is of primordial importance. Compost from different resources, now treated as wastes, could be one form of sustainable fertilizer creating a resilience of agriculture to the adverse effects of climate change. However, the safety of the produced compost regarding human pathogens, pharmaceuticals, and related resistance genes must be considered. We have assessed the effect of thermophilic composting of dry toilet contents, green cuttings, and straw, with and without biochar, on fecal indicators, the bacterial community, and antibiotic resistance genes (ARGs). Mature compost samples were analyzed regarding fecal indicator organisms, revealing low levels of Escherichia coli that are in line with German regulations for fertilizers. However, one finding of Salmonella spp. exceeded the threshold value. Cultivation of bacteria from the mature compost resulted in 200 isolates with 36.5% of biosafety level 2 (BSL-2) species. The majority is known as opportunistic pathogens that likewise occur in different environments. A quarter of the isolated BSL-2 strains exhibited multiresistance to different classes of antibiotics. Molecular analysis of total DNA before and after composting revealed changes in bacterial community composition and ARGs. 16S rRNA gene amplicon sequencing showed a decline of the two most abundant phyla Proteobacteria (start: 36–48%, end: 27–30%) and Firmicutes (start: 13–33%, end: 12–16%), whereas the abundance of Chloroflexi, Gemmatimonadetes, and Planctomycetes rose. Groups containing many human pathogens decreased during composting, like Pseudomonadales, Bacilli with Bacillus spp., or Staphylococcaceae and Enterococcaceae. Gene-specific PCR showed a decline in the number of detectable ARGs from 15 before to 8 after composting. The results reveal the importance of sufficiently high temperatures lasting for a sufficiently long period during the thermophilic phase of composting for reducing Salmonella to levels matching the criteria for fertilizers. However, most severe human pathogens that were targeted by isolation conditions were not detected. Cultivation-independent analyses also indicated a decline in bacterial orders comprising many pathogenic bacteria, as well as a decrease in ARGs. In summary, thermophilic composting could be a promising approach for producing hygienically safe organic fertilizer from ecological sanitation.

Metagenomic Insights Into the Changes of Antibiotic Resistance and Pathogenicity Factor Pools Upon Thermophilic Composting of Human Excreta

“…The phyla following in abundance accounted for less than 10% of the metagenomic reads ( Figure 2 ). The composition of the bacterial community was elucidated previously based on 16S rRNA gene amplicon sequencing ( Werner et al, 2022 ). The order of phyla (regarding their relative abundances) differed between amplicon and metagenomic data, but phyla with highest abundance were the same and general trends mostly corresponded.…”

“…Since bacterial community composition was assessed before ( Werner et al, 2022 ), here we focus on potential human pathogens. In Figure 3 the 40 most abundant genera and species with pathogenic potential are shown.…”

“…Cultivation-based assessment of indicator organisms for human pathogens (MPN technique for Salmonella and E. coli ) was described in Werner et al (2022) . E. coli MPNs were found to be below the German threshold values for fertilizers, whereas Salmonella was detected in one of the four compost piles not matching German regulations for fertilizer.…”

“…The composting trial (including feedstock material properties, composition and temperature measurements), sampling and sample pretreatment were described in detail in Werner et al (2022) . In brief, thermophilic windrow composting of dry toilet contents, green cuttings, straw, and urine, with and without biochar, was conducted in Eckernförde, Germany, between September 2018 and January 2019.…”

“…qPCR analyses were conducted on selected genes conferring resistance to different classes of antibiotics to quantify the gene concentrations in samples before, after 14-days (only repetition 1) and after composting. Genes were chosen based on previous PCR results of the samples ( aph(2″)-Ia , aph(3′)-IIIa , ermA , ermB , sul1 , sul2 , tetL and tetS ) ( Werner et al, 2022 ). qPCR was performed using a LightCycler ® 480 II instrument (Roche Diagnostics Ltd.) and the LightCycler ® 480 Probes Master (Roche Diagnostics Deutschland GmbH).…”