Microbial diversity in the municipal composting process and development of detection methods

Composting refers to aerobic degradation of organic material and is one of the main waste treatment methods used in Finland for treating separated organic waste. The composting process allows converting organic waste to a humus-like end product which can be used to increase the organic matter in agricultural soils, in gardening, or in landscaping. Microbes play a key role as degraders during the composting-process, and the microbiology of composting has been studied for decades, but there are still open questions regarding the microbiota in industrial composting processes. It is known that with the traditional, culturing-based methods only a small fraction, below 1%, of the species in a sample is normally detected. In recent years an immense diversity of bacteria, fungi and archaea has been found to occupy many different environments. Therefore the methods of characterising microbes constantly need to be developed further. In this thesis the presence of fungi and bacteria in full-scale and pilot-scale composting processes was characterised with cloning and sequencing. Several clone libraries were constructed and altogether nearly 6000 clones were sequenced. The microbial communities detected in this study were found to differ from the compost microbes observed in previous research with cultivation based methods or with molecular methods from processes of smaller scale, although there were similarities as well. The bacterial diversity was high. Based on the non-parametric coverage estimations, the number of bacterial operational taxonomic units (OTU) in certain stages of composting was over 500. Sequences similar to Lactobacillus and Acetobacteria were frequently detected in the early stages of drum composting. In tunnel stages of composting the bacterial community comprised of Bacillus, Thermoactinomyces, Actinobacteria and Lactobacillus. The fungal diversity was found to be high and phylotypes similar to yeasts were abundantly found in the full-scale drum and tunnel processes. In addition to phylotypes similar to Candida, Pichia and Geotrichum moulds from genus Thermomyces and Penicillium were observed in tunnel stages of composting. Zygomycetes were detected in the pilot-scale composting processes and in the compost piles. In some of the samples there were a few abundant phylotypes present in the clone libraries that masked the rare ones. The rare phylotypes were of interest and a method for collecting them from clone libraries for sequencing was developed. With negative selection of the abundant phylotyps the rare ones were picked from the clone libraries. Thus 41% of the clones in the studied clone libraries were sequenced. Since microbes play a central role in composting and in many other biotechnological processes, rapid methods for characterization of microbial diversity would be of value, both scientifically and commercially. Current methods, however, lack sensitivity and specificity and are therefore under development. Microarrays have been used in microbial ecology for a decade to study the presence or absence of certain microbes of interest in a multiplex manner. The sequence database collected in this thesis was used as basis for probe design and microarray development. The enzyme assisted detection method, ligation-detection-reaction (LDR) based microarray, was adapted for species-level detection of microbes characteristic of each stage of the composting process. With the use of a specially designed control probe it was established that a species specific probe can detect target DNA representing as little as 0.04% of total DNA in a sample. The developed microarray can be used to monitor composting processes or the hygienisation of the compost end product. A large compost microbe sequence dataset was collected and analysed in this thesis. The results provide valuable information on microbial community composition during industrial scale composting processes. The microarray method was developed based on the sequence database collected in this study. The method can be utilised in following the fate of interesting microbes during composting process in an extremely sensitive and specific manner. The platform for the microarray is universal and the method can easily be adapted for studying microbes from environments other than compost.Kompostoinnilla tarkoitetaan biologisesti hajoavan orgaanisen jätteen hajotusta ja se on yksi tärkeimmistä biojätteen käsittelytavoista Suomessa. Mullan kaltaista lopputuotetta voidaan käyttää lannoitteena maataloudessa ja puutarhanhoidossa sekä materiaalina viherrakentamisessa ja maisemoinnissa. Täten jätteenkäsittelymenetelmän ohella kompostointi on myös kierrätysmenetelmä. Kompostointiprosessissa mikrobit ovat tärkeässä roolissa orgaanisen aineen hajottajina. Vaikka kompostoinnin mikrobiologiaa onkin tutkittu jo vuosikymmeniä, mikrobisto teollisessa suuren mittakaavan kompostointilaitoksessa on suurelta osin tuntematon. Tässä väitöskirjatyössä selvitettiin sienten ja bakteerien esiintymistä teollisessa kompostointilaitoksessa ja pienemmässä tutkimuskäyttöön rakennetussa kompostointirummussa molekyylibiologisin menetelmin. Aiemmin on havaittu, että maljakasvatuksiin perustuvilla menetelmillä saadaan selville vain noin 1 % näytteen mikrobiyhteisöstä. Yli 6000 mikrobien tunnistusalueiden DNA-fragmenttia sekvensoitiin ja tuloksena oli yli 500 erilaista bakteerien tunnistussekvenssiä ja 127 erilaista sienten sekvenssiä. Näitä sekvenssejä verrattiin kansainvälisiin sekvenssitietokantoihin. Sekä bakteerien että sienten havaittiin eroavan aiemmissa tutkimuksissa löydetyistä mikrobeista, vaikka yhteneväisyyksiäkin löytyi. Myös aiemmin tunnistamattomia mikrobisekvenssejä oli runsaasti. Tutkimuksessa havaittiin, että mikrobiyhteisöt teollisen- ja tutkimusmittakaavan laitoksella erosivat selvästi. Kloonikirjastoissa on usein monta kappaletta samaa sekvenssiä. Väitöskirjatyössä kehitettiin menetelmä, jolla runsaslukuiset sekvenssit merkitään ennen sekvensointia ja vain harvalukuiset sekvensoidaan. Tällöin tietyt yleiset sekvenssityypit eivät estä harvalukuisempien sekvenssityyppien havainnointia. Menetelmän avulla havaittiin, että 41 % kloonikirjastojen klooneista oli harvinaisia ja vain ne sekvensoitiin. Säästö sekvensoinnissa ja käsiteltävän datan määrässä oli täten huomattava. Kompostin mikrobien esiintymisessä havaittiin eroja prosessin eri vaiheiden välillä. Koska kaikkien mikrobien kloonaaminen ja sekvensointi on aikaa vievää ja kiinnostuksen kohteena on usein vain muutama sekvenssityyppi, kehitettiin näiden sekvenssien havainnointia varten mikrosirudiagnostiikkaan perustuva menetelmä. Mikrosiruja on käytetty lajiston selvittämiseen jo vuosikymmeniä, mutta käytetyt menetelmät ovat olleet puutteellisia herkkyydeltään ja tarkkuudeltaan. Väitöskirjassa kehitetyn mikrosirumenetelmän avulla saavutettiin tarkkuus, jolla kaksi lähisukuista mikrobilajia voitiin erottaa toisistaan. Menetelmä perustuu kahden DNA-koettimen liittämiseen ja työssä suunnitellun kontrollikoettimen käyttöön. Menetelmä osoittautui hyvin herkäksi ja myös pieninä määrinä esiintyvät mikrobit voitiin havainnoida. Mikrosirua voidaan siksi hyödyntää kompostointiprosessin seuraamisessa tai kompostin lopputuotteen hygieenisyyden varmentamisessa. Tämän väitöskirjatyön tulokset tuovat tärkeää tietoa teollisen kompostointiprosessin mikrobiyhteisöstä. Suunniteltua mikrosirumenetelmää voidaan soveltaa myös muiden ympäristöjen mikrobien tutkimiseen

Candidatus Nitrosopolaris, a genus of putative ammonia-oxidizing archaea with a polar/alpine distribution

Ammonia-oxidizing archaea (AOA) are key players in the nitrogen cycle of polar soils. Here, we analyzed metagenomic data from tundra soils in Rásttigáisá, Norway, and recovered four metagenome-assembled genomes (MAGs) assigned to the genus ‘UBA10452’, an uncultured lineage of putative AOA in the order Nitrososphaerales (‘terrestrial group I.1b’), phylum Thaumarchaeota. Analysis of other eight previously reported MAGs and publicly available amplicon sequencing data revealed that the UBA10452 lineage is predominantly found in acidic polar and alpine soils. In particular, UBA10452 MAGs were more abundant in highly oligotrophic environments such as mineral permafrost than in more nutrient-rich, vegetated tundra soils. UBA10452 MAGs harbour multiple copies of genes related to cold tolerance, particularly genes involved in DNA replication and repair. Based on the phylogenetic, biogeographic, and ecological characteristics of 12 UBA10452 MAGs, which include a high-quality MAG (90.8% complete, 3.9% redundant) with a nearly complete 16S rRNA gene, we propose a novel Candidatus genus, Ca. Nitrosopolaris, with four species representing clear biogeographic/habitat clusters.Peer reviewe

Application of hybridization control probe to increase accuracy on ligation detection or minisequencing diagnostic microarrays

<p>Abstract</p> <p>Background</p> <p>Nucleic acid detection based on ligation reaction or single nucleotide extension of ssDNA probes followed by tag microarray hybridization provides an accurate and sensitive detection tool for various diagnostic purposes. Since microarray quality is crucial for reliable detection, these methods can benefit from correcting for microarray artefacts using specifically adapted techniques.</p> <p>Findings</p> <p>Here we demonstrate the application of a per-spot hybridization control oligonucleotide probe and a novel way of computing normalization for tag array data. The method takes into account the absolute value of the detection probe signal and the variability in the control probe signal to significantly alleviate problems caused by artefacts and noise on low quality microarrays.</p> <p>Conclusions</p> <p>Diagnostic microarray platforms require experimental and computational tools to enable efficient correction of array artefacts. The techniques presented here improve the signal to noise ratio and help in determining true positives with better statistical significance and in allowing the use of arrays with poor quality that would otherwise be discarded.</p

Growth and metabolic characteristics of fastidious meat-derived Lactobacillus algidus strains

Lactobacillus algidus is a meat spoilage bacterium often dominating the bacterial communities on chilled, packaged meat. Yet, L. algidus strains are rarely recovered from meat, and only few studies have focused on this species. The main reason limiting detailed studies on L. algidus is related to its poor growth on the media routinely used for culturing food spoilage bacteria. Thus, our study sought to develop reliable culture media for L. algidus to enable its recovery from meat, and to allow subculturing and phenotypic analyses of the strains. We assessed the growth of meat-derived L. algidus strains on common culture media and their modifications, and explored the suitability of potential media for the recovery of L. algidus from meat. Moreover, we determined whether 12 meat-derived L. algidus strains selected from our culture collection produce biogenic amines that may compromise safety or quality of meat, and finally, sequenced de novo and annotated the genomes of two meat-derived L. algidus strains to uncover genes and metabolic pathways relevant for phenotypic traits observed. MRS agar supplemented with complex substances (peptone, meat and yeast extract, liver digest) supported the growth of L. algidus, and allowed the recovery of new L. algidus isolates from meat. However, most strains grew poorly on standard MRS agar and on general-purpose media. In MRS broth, most strains grew well but a subset of strains required supplementation of MRS broth with additional cysteine. Supplementation of MRS broth with catalase allowed growth in aerated cultures suggesting that the strains produced hydrogen peroxide when grown aerobically. The strains tested (n = 12) produced ornithine from arginine and putrescine from agmatine, and two strains produced tyramine from tyrosine. Our findings reveal that L. algidus populations are underestimated if routine culture protocols are applied, and prompt concerns that L. algidus may generate tyramine or putrescine in meat or fermented meat products.Peer reviewe

Universal ligation-detection-reaction microarray applied for compost microbes

<p>Abstract</p> <p>Background</p> <p>Composting is one of the methods utilised in recycling organic communal waste. The composting process is dependent on aerobic microbial activity and proceeds through a succession of different phases each dominated by certain microorganisms. In this study, a ligation-detection-reaction (LDR) based microarray method was adapted for species-level detection of compost microbes characteristic of each stage of the composting process. LDR utilises the specificity of the ligase enzyme to covalently join two adjacently hybridised probes. A zip-oligo is attached to the 3'-end of one probe and fluorescent label to the 5'-end of the other probe. Upon ligation, the probes are combined in the same molecule and can be detected in a specific location on a universal microarray with complementary zip-oligos enabling equivalent hybridisation conditions for all probes. The method was applied to samples from Nordic composting facilities after testing and optimisation with fungal pure cultures and environmental clones.</p> <p>Results</p> <p>Probes targeted for fungi were able to detect 0.1 fmol of target ribosomal PCR product in an artificial reaction mixture containing 100 ng competing fungal ribosomal internal transcribed spacer (ITS) area or herring sperm DNA. The detection level was therefore approximately 0.04% of total DNA. Clone libraries were constructed from eight compost samples. The LDR microarray results were in concordance with the clone library sequencing results. In addition a control probe was used to monitor the per-spot hybridisation efficiency on the array.</p> <p>Conclusion</p> <p>This study demonstrates that the LDR microarray method is capable of sensitive and accurate species-level detection from a complex microbial community. The method can detect key species from compost samples, making it a basis for a tool for compost process monitoring in industrial facilities.</p

Pangenome and genomic taxonomy analyses of Leuconostoc gelidum and Leuconostoc gasicomitatum

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Host range of antibiotic resistance genes in wastewater treatment plant influent and effluent

10.1093/femsec/fiy038Wastewater treatment plants (WWTPs) collect wastewater from various sources for a multi-step treatment process. By mixing a large variety of bacteria and promoting their proximity, WWTPs constitute potential hotspots for the emergence of antibiotic resistant bacteria. Concerns have been expressed regarding the potential of WWTPs to spread antibiotic resistance genes (ARGs) from environmental reservoirs to human pathogens. We utilized epicPCR (Emulsion, Paired Isolation and Concatenation PCR) to detect the bacterial hosts of ARGs in two WWTPs. We identified the host distribution of four resistance-associated genes (tetM, int1, qacE Delta 1 and bla(OXA-58)) in influent and effluent. The bacterial hosts of these resistance genes varied between the WWTP influent and effluent, with a generally decreasing host range in the effluent. Through 16S rRNA gene sequencing, it was determined that the resistance gene carrying bacteria include both abundant and rare taxa. Our results suggest that the studied WWTPs mostly succeed in decreasing the host range of the resistance genes during the treatment process. Still, there were instances where effluent contained resistance genes in bacterial groups not carrying these genes in the influent. By permitting exhaustive profiling of resistance-associated gene hosts in WWTP bacterial communities, the application of epicPCR provides a new level of precision to our resistance gene risk estimates.Peer reviewe

Ecology determines how low antibiotic concentration impacts community composition and horizontal transfer of resistance genes

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Microbiomes in the Context of Refrigerated Raw Meat Spoilage

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Complete genome sequence of Leuconostoc gelidum subsp. gasicomitatum KG16-1, isolated from vacuum-packaged vegetable sausages

Leuconostoc gelidum subsp. gasicomitatum is a predominant lactic acid bacterium (LAB) in spoilage microbial communities of different kinds of modified-atmosphere packaged (MAP) food products. So far, only one genome sequence of a poultry-originating type strain of this bacterium (LMG 18811T) has been available. In the current study, we present the completely sequenced and functionally annotated genome of strain KG16-1 isolated from a vegetable-based product. In addition, six other vegetable-associated strains were sequenced to study possible “niche” specificity suggested by recent multilocus sequence typing. The genome of strain KG16-1 consisted of one circular chromosome and three plasmids, which together contained 2,035 CDSs. The chromosome carried at least three prophage regions and one of the plasmids encoded a galactan degradation cluster, which might provide a survival advantage in plant-related environments. The genome comparison with LMG 18811T and six other vegetable strains suggests no major differences between the meat- and vegetable-associated strains that would explain their “niche” specificity. Finally, the comparison with the genomes of other leuconostocs highlights the distribution of functionally interesting genes across the L. gelidum strains and the genus Leuconostoc.Peer reviewe