Microbial community structure and dynamics in thermophilic composting viewed through metagenomics and metatranscriptomics

Composting is a promising source of new organisms and thermostable enzymes that may be helpful in environmental management and industrial processes. Here we present results of metagenomicand metatranscriptomic-based analyses of a large composting operation in the Sao Paulo Zoo Park. This composting exhibits a sustained thermophilic profile (50 degrees C to 75 degrees C), which seems to preclude fungal activity. The main novelty of our study is the combination of time-series sampling with shotgun DNA, 16S rRNA gene amplicon, and metatranscriptome high-throughput sequencing, enabling an unprecedented detailed view of microbial community structure, dynamics, and function in this ecosystem. The time-series data showed that the turning procedure has a strong impact on the compost microbiota, restoring to a certain extent the population profile seen at the beginning of the processand that lignocellulosic biomass deconstruction occurs synergistically and sequentially, with hemicellulose being degraded preferentially to cellulose and lignin. Moreover, our sequencing data allowed near-complete genome reconstruction of five bacterial species previously found in biomass-degrading environments and of a novel biodegrading bacterial species, likely a new genus in the order Bacillales. The data and analyses provided are a rich source for additional investigations of thermophilic composting microbiology.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Provost's Office for Research of the University of Sao PauloCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Sao Paulo, Inst Quim, Dept Bioquim, Sao Paulo, BrazilUniv Sao Paulo, Programa Pos Graduacao Interunidades Bioinformat, Sao Paulo, BrazilUniv Sao Paulo, Escola Artes Ciencias & Humanidades, Sao Paulo, Brazil|Fundacao Parque Zool Sao Paulo, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Ciencias Biol, Sao Paulo, BrazilBiocomplex Inst Virginia, Blacksburg, VA USADepartamento de Ciências Biológicas, Universidade Federal de São Paulo, São Paulo, BrazilFAPESP: 2011/50870-6Web of Scienc

Metagenomic analysis of a tropical composting operation at the São Paulo Zoo Park reveals diversity of biomass degradation functions and organisms.

Composting operations are a rich source for prospection of biomass degradation enzymes. We have analyzed the microbiomes of two composting samples collected in a facility inside the Sao Paulo Zoo Park, in Brazil. All organic waste produced in the park is processed in this facility, at a rate of four tons/day. Total DNA was extracted and sequenced with Roche/454 technology, generating about 3 million reads per sample. To our knowledge this work is the first report of a composting whole-microbial community using high-throughput sequencing and analysis. The phylogenetic profiles of the two microbiomes analyzed are quite different, with a clear dominance of members of the Lactobacillus genus in one of them. We found a general agreement of the distribution of functional categories in the Zoo compost metagenomes compared with seven selected public metagenomes of biomass deconstruction environments, indicating the potential for different bacterial communities to provide alternative mechanisms for the same functional purposes. Our results indicate that biomass degradation in this composting process, including deconstruction of recalcitrant lignocellulose, is fully performed by bacterial enzymes, most likely by members of the Clostridiales and Actinomycetales orders.FAPESP 2009/52030-5RCNPqCAPE

Metagenomics and metatranscriptomics of the São Paulo Zoo Park composting microbiota

As compostagens abrigam uma grande riqueza microbiológica, englobando populações com distintos requerimentos e tolerâncias fisiológicas que se sucedem ao longo do processo de biodegradação aeróbica da matéria orgânica e que resultam na elevação espontânea de temperatura até 80° C. Com a utilização de abordagens de metagenômica e metatranscritômica, investigamos a composição e a diversidade taxonômica, bem como as funções metabólicas de comunidades microbianas da compostagem termofílica do Parque Zoológico de São Paulo. Foram analisadas amostras em série temporal de duas composteiras (ZC3 e ZC4), as quais exibiram temperaturas entre 50ºC-75ºC ao longo de 99 dias do processo. Verificamos que a degradação de toda a biomassa foi realizada essencialmente por bactérias, e que a estrutura e composição das comunidades microbianas variam ao longo do processo, com elevada abundância relativa das Ordens Clostridiales, Bacillales e Actinomycetales, assim como observado em outros sistemas de compostagem. Entre os organismos abundantes no processo, identificamos unidades taxonômicas operacionais (OTUs) referentes a organismos não-cultiváveis e/ou com genoma ainda desconhecido. O genoma parcial de uma destas OTUs foi reconstruído, a qual provavelmente pertence a um novo gênero da ordem Bacillales. A dinâmica do processo de compostagem foi evidenciada pela variação do número de OTUs e do índice de diversidade filogenética ao longo do tempo, sendo que o início do processo e a fase após a revira apresentaram a maior diversidade. Os resultados indicam que o processo de revira (aeração da massa de composto) impacta fortemente a estrutura e a composição da microbiota e que a desconstrução da biomassa vegetal ocorre de forma sinérgica e sequencial. A variedade de microrganismos e de funções metabólicas ativas na compostagem termofílica reforça o seu potencial de ser uma promissora fonte de bactérias e enzimas termorresistentes úteis em processos industriais.Composting harbors considerable microbial richness, comprising populations with distinct physiological requirements and tolerances that succeed one another throughout the aerobic biodegradation of the organic matter, resulting in spontaneous temperature rise up to 80° C. Using metagenomic- and metatranscritomic-based approaches, we investigated the composition and taxonomic diversity as well as metabolic functions of microbial communities of a thermophilic composting operation in the São Paulo Zoo Park. We have analyzed time-series samples from two composting cells (ZC3 and ZC4) which exhibited sustained thermophilic profile (50°C-75°C) over 99 days of the process. We found that all biomass degradation was essentially performed by bacteria. The structure and composition of microbial communities vary throughout the process with a high relative abundance of Clostridiales, Bacillales and Actinomycetales, as observed in other composting systems. Among the organisms abundant in the process, we identify Operational Taxonomic Units (OTUs) of uncultivated organisms or with unknown genomes. The partial genome of one of these OTUs was obtained and shown to belong probably to a new genus of Bacillales. Our time-series data showed that the number of OTUs and phylogenetic diversity index changed during composting revealing the dynamics of the process, with the beginning and the stage after turning procedure presenting the highest diverse microbiota. These results indicate that the turning procedure (compost aeration) strongly impacts the microbiota structure and composition and that the deconstruction of the biomass occurs synergistically and sequentially. The huge diversity of microorganisms and metabolic functions active in thermophilic composting strengthen its potential as a promising source of new bacteria and thermostable enzymes that may be helpful in industrial processes

Three novel Pseudomonas phages isolated from composting provide insights into the evolution and diversity of tailed phages

Abstract Background Among viruses, bacteriophages are a group of special interest due to their capacity of infecting bacteria that are important for biotechnology and human health. Composting is a microbial-driven process in which complex organic matter is converted into humus-like substances. In thermophilic composting, the degradation activity is carried out primarily by bacteria and little is known about the presence and role of bacteriophages in this process. Results Using Pseudomonas aeruginosa as host, we isolated three new phages from a composting operation at the Sao Paulo Zoo Park (Brazil). One of the isolated phages is similar to Pseudomonas phage Ab18 and belongs to the Siphoviridae YuA-like viral genus. The other two isolated phages are similar to each other and present genomes sharing low similarity with phage genomes in public databases; we therefore hypothesize that they belong to a new genus in the Podoviridae family. Detailed genomic descriptions and comparisons of the three phages are presented, as well as two new clusters of phage genomes in the Viral Orthologous Clusters database of large DNA viruses. We found sequences encoding homing endonucleases that disrupt a putative ribonucleotide reductase gene and an RNA polymerase subunit 2 gene in two of the phages. These findings provide insights about the evolution of two-subunits RNA polymerases and the possible role of homing endonucleases in this process. Infection tests on 30 different strains of bacteria reveal a narrow host range for the three phages, restricted to P. aeruginosa PA14 and three other P. aeruginosa clinical isolates. Biofilm dissolution assays suggest that these phages could be promising antimicrobial agents against P. aeruginosa PA14 infections. Analyses on composting metagenomic and metatranscriptomic data indicate association between abundance variations in both phage and host populations in the environment. Conclusion The results about the newly discovered and described phages contribute to the understanding of tailed bacteriophage diversity, evolution, and role in the complex composting environment

Relative abundance of COG functional categories for ZC1 and ZC2 and seven public metagenomes.

<p>Assembled sequence reads were classified into the 25 COG categories designated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061928#pone-0061928-g005" target="_blank">Figure 5</a> and their relative abundances for each metagenome were estimated considering the respective total number of protein coding sequences with function prediction. The public metagenomes included in the comparison are benzene-degrading bioreactor, biofuel reactor, compost minireactor, termite hindgut, poplar biomass bioreactor, lake sediment and soil rain forest, whose features are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061928#pone.0061928.s008" target="_blank">Table S5</a>. Asterisks indicate statistically significant values.</p

Relative abundance of COG functional categories for ZC1 and ZC2 metagenomes.

<p>Assembled sequence reads were classified into the 25 COG functional categories, and their relative abundances for ZC1 and ZC2 metagenomes were estimated considering the total number of protein coding sequences with function prediction. Designations of functional categories: A: RNA processing and modification, B: Chromatin structure and dynamics, C: Energy production and conversion, D: Cell cycle control, cell division, chromosome partitioning, E: Amino acid transport and metabolism, F: Nucleotide transport and metabolism, G: Carbohydrate transport and metabolism, H: Coenzyme transport and metabolism, I: Lipid transport and metabolism, J: Translation, ribosomal structure and biogenesis, K: Transcription, L: Replication, recombination and repair, M: Cell wall/membrane/envelope biogenesis, N: Cell motility, O: Posttranslational modification, protein turnover, chaperones, P: Inorganic ion transport and metabolism, Q: Secondary metabolites biosynthesis, transport and catabolism, R: General function prediction only, S: Function unknown, T: Signal transduction mechanisms, U: Intracellular trafficking, secretion, and vesicular transport, V: Defense mechanisms, W: Extracellular structures, Y: Nuclear structure, Z: Cytoskeleton.</p

454 GS FLX Titanium pyrosequencing and Newbler assembly metrics of two metagenomic DNA samples from São Paulo Zoo composting.

<p>454 GS FLX Titanium pyrosequencing and Newbler assembly metrics of two metagenomic DNA samples from São Paulo Zoo composting.</p

ZC1 large contig encoding pectin degradation enzymes.

<p>ZC1 sequences assembled into a 27,919 bp contig encoding the following proteins: 1. Beta-xylosidase (376 aa, COG3507); 2. Dehydrogenases (280 aa, COG1028); 3. hypothetical protein (379 aa); 4. hypothetical protein (283 aa); 5. 5-keto 4-deoxyuronate isomerase (280 aa, COG3717); 6. Dehydrogenases (267 aa, COG1028);7. hypothetical protein (1799 aa); 8. SusD family protein (606 aa, pfam07980); 9. TonB-linked outer membrane protein (1068 aa, COG4771); 10. Pectate lyase (518 aa, COG3866); 11. Predicted unsaturated glucuronyl hydrolase (398 aa, COG4225); 12. Pectin methylesterase (568 aa, COG4677); 13. Endopolygalacturonase (523 aa, COG5434); 14. Nucleoside-diphosphate-sugar epimerase (326 aa, COG0451); 15. Nucleoside-diphosphate-sugar pyrophosphorylase (249 aa, pfam00483); 16. Galactokinase (377 aa, COG0153); 17. Soluble lytic murein transglycosylase (347 aa, COG0741); 18. hypothetical protein (235 aa); 19. Predicted UDP-glucose 6-dehydrogenase (283 aa, COG1004).</p