Adam Pyzik

The scheme illustrates a droplet microfluidic workflow for isolating microorganisms using high-throughput screening (HTS), in which individual cells are encapsulated in droplets, cultivated, sorted, and ultimately recovered as selected strains.

Screening microbial diversity is critical for discovering novel strains with enhanced biocatalytic capabilities. Traditional cultivation techniques often miss rare or slow-growing microorganisms, limiting our understanding of microbial biodiversity and its practical applications. Droplet-based microfluidics has emerged as a powerful platform for ultra-high- throughput screening of single cells or microbial consortia, encapsulated within microscale droplets that act as precisely controlled bioreactors. This tutorial review focuses on the application of droplet microfluidics for microbial cultivation and isolation, emphasizing advantages such as rapid throughput and the ability to isolate and analyze individual cells. Unlike previous reviews that broadly address microfluidic technologies or target specific applications like antibiotic resistance or enzyme discovery, we concentrate on protocols specifically designed for the clonal and parallel cultivation of microorganisms from environmental samples. Droplet microfluidics has already shown significant promise in environmental biotechnology, bioremediation, and microbial ecology. Its broader adoption dramatically reduces assay volumes and costs while enhancing screening efficiency, positioning droplet microfluidics as a key technology for the future of environmental microbiology research.

Cultural heritage objects constitute a very diverse environment, inhabited by various bacteria and fungi. The impact of these microorganisms on the degradation of artworks is undeniable, but at the same time, some of them may be applied for the efficient biotreatment of cultural heritage assets. Interventions with microorganisms have been proven to be useful in restoration of artworks, when classical chemical and mechanical methods fail or produce poor or short-term effects. The path to understanding the impact of microbes on historical objects relies mostly on multidisciplinary approaches, combining novel meta-omic technologies with classical cultivation experiments, and physico-chemical characterization of artworks. In particular, the development of metabolomic- and metatranscriptomic-based analyses associated with metagenomic studies may significantly increase our understanding of the microbial processes occurring on different materials and under various environmental conditions. Moreover, the progress in environmental microbiology and biotechnology may enable more effective application of microorganisms in the biotreatment of historical objects, creating an alternative to highly invasive chemical and mechanical methods.

Biodeterioration is a serious threat to cultural heritage objects and buildings. The deterioration of a given material often incurs irreparable losses in terms of uniqueness and historical value. Hence preventive actions should be taken. One important challenge is to identify microbes involved in the biodeterioration process. In this study, we analyzed the microbial diversity of an ancient architectonical structure of the Rotunda of Sts. Felix and Adauctus, which is a part of the Wawel Royal Castle located in Krakow, Poland. The Rotunda is unavailable to tourists and could be treated as an extreme habitat due to the low content of nutrients coming either from sandstone plates bound with lime mortar or air movement. Microbial diversity was analyzed with the use of the high-throughput sequencing of marker genes corresponding to fragments of 16S rDNA (for Bacteria) and ITS2 (internal transcribed spacer 2) (for Fungi). The results showed that the microbial community adhered to wall surfaces is, to a large extent, endemic. Furthermore, alongside many microorganisms that could be destructive to masonry and mortar (e.g., Pseudomonas, Aspergillus), there were also bacteria, such as species of genera Bacillus, Paenisporosarcina, and Amycolatopsis, that can positively affect wall surface properties by reducing the damage caused by the presence of other microorganisms. We also showed that airborne microorganisms probably have little impact on the biodeterioration process as their abundance in the microbial community adhered to the ancient walls was very low.

Although interactions between microorganisms involved in biogas production are largely uncharted, it is commonly accepted that methanogenic Archaea are essential for the process. Methanogens thrive in various environments, but the most extensively studied communities come from biogas plants. In this study, we employed a metagenomic analysis of deeply sequenced methanogenic communities, which allowed for comparison of taxonomic and functional diversity as well as identification of microorganisms directly involved in various stages of methanogenesis pathways. A comprehensive metagenomic approach was used to compare seven environmental communities, originating from an agricultural biogas plant, cattle-associated samples, a lowland bog, sewage sludge from a wastewater treatment plant and sediments from an ancient gold mine. In addition to the native consortia, two laboratory communities cultivated on maize silage as the sole substrate were also analyzed. Results showed that all anaerobic communities harbored genes of all known methanogenesis pathways, but their abundance varied greatly between environments and that genes were encoded by different methanogens. Identification of microorganisms directly involved in different stages of methane production revealed that hydrogenotrophic methanogens, such as Methanoculleus, Methanobacterium, Methanobrevibacter, Methanocorpusculum or Methanoregula, predominated in most native communities, whereas acetoclastic Methanosaeta seemed to be the key methanogen in the wastewater treatment plant. Furthermore, in many environments, the methylotrophic pathway carried out by representatives of Methanomassiliicoccales, such as Candidatus Methanomethylophilus and Candidatus Methanoplasma, seemed to play an important role in methane production. In contrast, in stable laboratory reactors substrate versatile Methanosarcina predominated. The metagenomic approach presented in this study allowed for deep exploration and comparison of nine environments in which methane production occurs. Different abundance of methanogenesis-related functions was observed and the functions were analyzed in the phylogenetic context in order to identify microbes directly involved in methane production. In addition, a comparison of two metagenomic analytical tools, MG-RAST and MetAnnotate, revealed that combination of both allows for a precise characterization of methanogenic communities.

A well-balanced microbial consortium is crucial for efficient biogas production. In turn, one of a major factor that influence on the structure of anaerobic digestion consortium is a source of microorganisms which are used as an inoculum. This study evaluated the influence of inoculum sources (with various origin) on adaptation of a biogas community and the efficiency of the biomethanization of maize silage. As initial inocula for anaerobic digestion of maize silage the samples from: (i) an agricultural biogas plant (ABP) which utilizes maize silage as a main substrate, (ii) cattle slurry (CS), which contain elevated levels of lignocelluloses materials, and (iii) raw sewage sludge (RSS) with low content of plant origin materials were used. The adaptation of methanogenic consortia was monitored during a series of passages, and the functionality of the adapted consortia was verified through start-up operation of anaerobic digestion in two-stage reactors. During the first stages of the adaptation phase, methanogenic consortia occurred very slowly, and only after several passages did the microbial community adapts to allow production of biogas with high methane content. The ABP consortium revealed highest biogas production in the adaptation and in the start-up process. The biodiversity dynamics monitored during adaptation and start-up process showed that community profile changed in a similar direction in three studied consortia. Native communities were very distinct to each other, while at the end of the Phase II of the start-up process microbial diversity profile was similar in all consortia. All adopted bacterial communities were dominated by representatives of Porphyromonadaceae, Rikenellaceae, Ruminococcaceae and Synergistaceae. A shift from low acetate-preferring acetoclastic Methanosaetaceae (ABP and RSS) and/or hydrogenotrophic Archaeae, e.g. Methanomicrobiaceae (CS) prevailing in the inoculum samples to larger populations of high acetate-preferring acetoclastic Methanosarcinaceae was observed by the end of the experiment. As a result, three independent, functional communities that syntrophically produced methane from acetate (primarily) and H2/CO2, methanol and methylamines were adapted. This study provides new insights into the specific process by which different inocula sampled from typical methanogenic environments that are commonly used to initiate industrial installations gradually adapted to allow biogas production from maize silage.

Methanogenic Archaea produce approximately one billion tons of methane annually, but their biology remains largely unknown. This is partially due to the large phylogenetic and phenotypic diversity of this group of organisms, which inhabit various anoxic environments including peatlands, freshwater sediments, landfills, anaerobic digesters and the intestinal tracts of ruminants. Research is also hampered by the inability to cultivate methanogenic Archaea. Therefore, biodiversity studies have relied on the use of 16S rRNA and mcrA [encoding the α subunit of the methyl coenzyme M (methyl-CoM) reductase] genes as molecular markers for the detection and phylogenetic analysis of methanogens. Here, we describe four novel molecular markers that should prove useful in the detailed analysis of methanogenic consortia, with a special focus on methylotrophic methanogens. We have developed and validated sets of degenerate PCR primers for the amplification of genes encoding key enzymes involved in methanogenesis: mcrB and mcrG (encoding β and γ subunits of the methyl-CoM reductase, involved in the conversion of methyl-CoM to methane), mtaB (encoding methanol-5-hydroxybenzimidazolylcobamide Co-methyltransferase, catalyzing the conversion of methanol to methyl-CoM) and mtbA (encoding methylated [methylamine-specific corrinoid protein]:coenzyme M methyltransferase, involved in the conversion of mono-, di- and trimethylamine into methyl-CoM). The sensitivity of these primers was verified by high-throughput sequencing of PCR products amplified from DNA isolated from microorganisms present in anaerobic digesters. The selectivity of the markers was analyzed using phylogenetic methods. Our results indicate that the selected markers and the PCR primer sets can be used as specific tools for in-depth diversity analyses of methanogenic consortia.