Culture-Independent Analysis of Linuron-Mineralizing Microbiota and Functions in on-Farm Biopurification Systems via DNA-Stable Isotope Probing: Comparison with Enrichment Culture.

Our understanding of the microorganisms involved in in situ biodegradation of xenobiotics, like pesticides, in natural and engineered environments is poor. On-farm biopurification systems (BPSs) treat farm-produced pesticide-contaminated wastewater to reduce surface water pollution. BPSs are a labor and cost-efficient technology but are still mainly operated as black box systems. We used DNA-stable isotope probing (DNA-SIP) and classical enrichment to be informed about the organisms responsible for in situ degradation of the phenylurea herbicide linuron in a BPS matrix. DNA-SIP identified Ramlibacter, Variovorax, and an unknown Comamonadaceae genus as the dominant linuron assimilators. While linuron-degrading Variovorax strains have been isolated repeatedly, Ramlibacter has never been associated before with linuron degradation. Genes and mobile genetic elements (MGEs) previously linked to linuron catabolism were enriched in the heavy DNA-SIP fractions, suggesting their involvement in in situ linuron assimilation. BPS material free cultivation of linuron degraders from the same BPS matrix resulted in a community dominated by Variovorax, while Ramlibacter was not observed. Our study provides evidence for the role of Variovorax in in situ linuron biodegradation in a BPS, alongside other organisms like Ramlibacter, and further shows that cultivation results in a biased representation of the in situ linuron-assimilating bacterial populations.

Biodegradable Mineral Plastics.

Mineral plastics are a promising class of bio-inspired materials that offer exceptional properties, like self-heal ability, stretchability in the hydrogel state, and high hardness, toughness, transparency, and non-flammability in the dry state along with reversible transformation into the hydrogel by addition of water. This enables easy reshape-ability and recycling like the solubility in mild acids to subsequently form mineral plastics again by base addition. However, current mineral plastics rely on petrochemistry, are hardly biodegradable, and thus persistent in nature. This work presents the next generation of mineral plastics, which are bio-based and biodegradable, making them a promising, new class of polymers for the development of environmentally friendly materials. Physically cross-linked (poly)glutamic-acid (PGlu)-based mineral plastics are synthesized using various alcohol-water mixtures, metal ion ratios and molecular weights. The rheological properties are easily adjusted using these parameters. The general procedure involves addition of equimolar solution of CaCl2 to PGlu in equal volumes followed by addition of iPrOH (iPrOH:H2 O = 1:1) under vigorous stirring conditions. The ready biodegradability of PGlu/CaFe mineral plastic is confirmed in this study where the elements N, Ca, and Fe present in it tend to act as additional nutrients, supporting the growth of microorganisms and consequently, promoting the biodegradation process.

The Transcription Factor Roc1 Is a Key Regulator of Cellulose Degradation in the Wood-Decaying Mushroom Schizophyllum commune.

Wood-decaying fungi of the class Agaricomycetes (phylum Basidiomycota) are saprotrophs that break down lignocellulose and play an important role in nutrient recycling. They secrete a wide range of extracellular plant cell wall degrading enzymes that break down cellulose, hemicellulose, and lignin, the main building blocks of plant biomass. Although the production of these enzymes is regulated mainly at the transcriptional level, no activating regulators have been identified in any wood-decaying fungus in the class Agaricomycetes. We studied the regulation of cellulase expression in the wood-decaying fungus Schizophyllum commune. Comparative genomics and transcriptomics on two wild isolates revealed a Zn2Cys6-type transcription factor gene (roc1) that was highly upregulated during growth on cellulose, compared to glucose. It is only conserved in the class Agaricomycetes. A roc1 knockout strain showed an inability to grow on medium with cellulose as sole carbon source, and growth on cellobiose and xylan (other components of wood) was inhibited. Growth on non-wood-related carbon sources was not inhibited. Cellulase gene expression and enzyme activity were reduced in the Δroc1 strain. ChIP-Seq identified 1474 binding sites of the Roc1 transcription factor. Promoters of genes involved in lignocellulose degradation were enriched with these binding sites, especially those of LPMO (lytic polysaccharide monooxygenase) CAZymes, indicating that Roc1 directly regulates these genes. A conserved motif was identified as the binding site of Roc1, which was confirmed by a functional promoter analysis. Together, Roc1 is a key regulator of cellulose degradation and the first identified in wood-decaying fungi in the phylum Basidiomycota. IMPORTANCE Wood-degrading fungi in the phylum Basidiomycota play a crucial role in nutrient recycling by breaking down all components of wood. Fungi have evolved transcriptional networks that regulate expression of wood-degrading enzymes, allowing them to prioritize one nutrient source over another. However, to date all these transcription factors have been identified in the phylum Ascomycota, which is only distantly related to the phylum Basidiomycota. Here, we identified the transcription factor Roc1 as a key regulator of cellulose degradation in the mushroom-forming and wood-degrading fungus Schizophyllum commune. Roc1 is highly conserved in the phylum Basidiomycota. Using comparative genomics, transcriptomics, ChIP-Seq and promoter analysis we have identified direct targets of Roc1, as well as other aspects of the transcriptional response to cellulose.

DNA-SIP and repeated isolation corroborate Variovorax as a key organism in maintaining the genetic memory for linuron biodegradation in an agricultural soil.

The frequent exposure of agricultural soils to pesticides can lead to microbial adaptation, including the development of dedicated microbial populations that utilize the pesticide compound as a carbon and energy source. Soil from an agricultural field in Halen (Belgium) with a history of linuron exposure has been studied for its linuron-degrading bacterial populations at two time points over the past decade and Variovorax was appointed as a key linuron degrader. Like most studies on pesticide degradation, these studies relied on isolates that were retrieved through bias-prone enrichment procedures and therefore might not represent the in situ active pesticide-degrading populations. In this study, we revisited the Halen field and applied, in addition to enrichment-based isolation, DNA stable isotope probing (DNA-SIP), to identify in situ linuron-degrading bacteria in linuron-exposed soil microcosms. Linuron dissipation was unambiguously linked to Variovorax and its linuron catabolic genes and might involve the synergistic cooperation between two species. Additionally, two novel linuron-mineralizing Variovorax isolates were obtained with high 16S rRNA gene sequence similarity to strains isolated from the same field a decade earlier. The results confirm Variovorax as a prime in situ degrader of linuron in the studied agricultural field soil and corroborate the genus as key for maintaining the genetic memory of linuron degradation functionality in that field.

Quantifying microwear on experimental Mistassini quartzite scrapers: preliminary results of exploratory research using LSCM and scale-sensitive fractal analysis.

Although previous use-wear studies involving quartz and quartzite have been undertaken by archaeologists, these are comparatively few in number. Moreover, there has been relatively little effort to quantify use-wear on stone tools made from quartzite. The purpose of this article is to determine the effectiveness of a measurement system, laser scanning confocal microscopy (LSCM), to document the surface roughness or texture of experimental Mistassini quartzite scrapers used on two different contact materials (fresh and dry deer hide). As in previous studies using LSCM on chert, flint, and obsidian, this exploratory study incorporates a mathematical algorithm that permits the discrimination of surface roughness based on comparisons at multiple scales. Specifically, we employ measures of relative area (RelA) coupled with the F-test to discriminate used from unused stone tool surfaces, as well as surfaces of quartzite scrapers used on dry and fresh deer hide. Our results further demonstrate the effect of raw material variation on use-wear formation and its documentation using LSCM and RelA.