Culture-based and culture-independent approach for the study of the methanogens and obligate anaerobes from different landfill sites

Abstract

The landfill is a cheap way of solid waste management in developing countries. The majority of landfills are non-sanitary and work as open garbage dumping sites and pose threats to public and environmental health. Therefore, an in-depth understanding of the chemistry and microbiology of landfills is imperative to develop the right policies for landfill management. In the current study, we investigated the chemistry and microbiology of three Indian landfill sites using culture-based and culture-independent molecular approaches. Our data indicate that the nature of landfills varies from site to site in terms of chemistry, pollutants, and pathogens. We also enriched and cultivated three methanogens using an optimized medium and constructed two high-quality draft genomes from enriched microbiomes using metagenome-assembled genome approaches. The phylogenomic study of one draft genome showed the highest 93% sequence similarity with members of Methanomassiliicoccaceae and was always enriched with Acholoplasma and Anaerohalosphaera lusitana. Despite all the efforts, we did not isolate it in pure culture and hypothesized that for the cultivation of some not-yet-cultured methanogen, the presence of other organisms plays an important role, and their syntrophic interaction must be discerned for its successful cultivation in the future. Co-cultivation of amino acid-degrading organisms indicates that their co-culture can assist in boosting the growth of methanogens. In addition, our data indicated that landfill leachate contains a heavy load of pollutants and treatment is a must before discharge in nature or use in irrigation or biofertilizer.

Keywords: anaerobes; contamination; landfill; leachate; methanogens.

FIGURE 1

Representation of ANI values of different strains of methanogens show close similarity with the genome obtained from this study.

FIGURE 2

Neighbor-joining phylogenetic tree construction based on 16S rRNA gene sequence. Selected sequences are labeled according to their habitats (landfill leachate sludge, mesophilic, thermophilic, diverse environment, freshwater, extremely acidophilic, termite gut, rumen gut, and human gut). Bootstrap support values are shown as a percentage before the respective nodes; the scale bar indicates the number of substitutions per site. Phylogenetic clusters are indicated on the right of the tree. Methanomassiliicoccus luminyensis 26 of tree is proposed “Candidatus Methanomassiliicoccus indica.”

FIGURE 3

Evolutionary relationships of methyl-coenzyme M reductase (McrA) of different methanogens. The evolutionary history was constructed using the neighbor-joining method. The analysis involved 21 amino acid sequences of mcrA genes. MEGA7 Kumar et al. (2016). Bootstrap support values are shown as a percentage before the respective nodes; the scale bar indicates the number of substitutions per site. Phylogenetic clusters are indicated on the right of the tree. Methanomassiliicoccus luminyensis 26 of tree is proposed “Candidatus Methanomassiliicoccus indica.”

FIGURE 4

Phylogenetic tree constructed using phylogenomic approach from draft genome sequence obtained from this study and its close relatives.

FIGURE 5

A putative illustration of syntrophic mechanism: Methanogens employ two separate pathways to convert CO₂ into methane: either the methyl group of acetate or the reduction of carbon dioxide with electrons from hydrogen or format. Hence, CO₂ and acetate are generated in the fermentation of glucose and glycogen. Biochemical pathway of H₂-dependent reduction of CO₂ to CH₄. The C1 moiety is transferred from CO₂ via MF, H₄MPT, and CoM (CoM-SH) into CH₄. Details of putative proteins of acetotrophic pathways in M. luminyensis 26. Electron-donating species formate is produced by the anaerobic degradation of organic matter by fermenting and syntrophic bacteria. Enzyme abbreviations are as follows: Fd, ferredoxin; HydABC, bifurcating [Fe-Fe] hydrogenase; HyaABC, [NiFe] hydrogenase; Acs, acetyl-coenzyme A synthetase; CooS, carbon monoxide dehydrogenase; CdhA, acetyl-CoA decarbonylase/synthase complex; Mtr, methyl-tetrahydromethanopterin: CoM methyltransferase; McrABG, methyl-coenzyme M reductase; HdrED, coenzyme B-coenzyme M heterodisulfide reductase; FpoABDHIJKLMNO, F₄₂₀H₂ dehydrogenase.