Transcriptome profiling of Paraburkholderia aromaticivorans AR20-38 during ferulic acid bioconversion.

The importance and need of renewable-based, sustainable feedstocks increased in recent years. Lignin-derived monomers have high potential, energetic and economic value in the microbial bioconversion to valuable biomolecules. The bacterium Paraburkholderia aromaticivorans AR20-38 produces a remarkable yield of vanillic acid from ferulic acid at moderate and low temperatures and is therefore a good candidate for biotechnological applications. To understand this bioconversion process on a molecular level, a transcriptomic study during the bioconversion process was conducted to elucidate gene expression patterns. Differentially expressed genes, cellular transporters as well as transcriptional factors involved in the bioconversion process could be described. Additional enzymes known for xenobiotic degradation were differentially expressed and a potential membrane vesicle mechanism was detected. The bioconversion mechanism on a transcriptional level of P. aromaticivorans could be elucidated and results can be used for strain optimization. Additionally, the transcriptome study showed the high potential of the strain for other degradation applications.

Low-Temperature Biodegradation of Lignin-Derived Aromatic Model Monomers by the Cold-Adapted Yeast Rhodosporidiobolus colostri Isolated from Alpine Forest Soil.

The contribution of cold-adapted yeasts to the emerging field of lignin biovalorization has not yet been studied. The red-pigmented basidiomycetous yeast strain Rhodosporidiobolus colostri DBVPG 10655 was examined for its potential to degrade five selected lignin-derived aromatic monomers (syringic acid, p-coumaric acid, 4-hydroxybenzoic acid, ferulic acid, and vanillic acid). The strain utilized p-coumaric acid, 4-hydroxybenzoic acid, and ferulic acid not only as the sole carbon source; full biodegradation occurred also in mixtures of multiple monomers. Vanillic acid was not utilized as the sole carbon source, but was degraded in the presence of p-coumaric acid, 4-hydroxybenzoic acid, and ferulic acid. Syringic acid was utilized neither as the sole carbon source nor in mixtures of compounds. Biodegradation of lignin-derived aromatic monomers was detected over a broad temperature range (1-25 °C), which is of ecological significance and of biotechnological relevance.

Biodegradation Potential and Putative Catabolic Genes of Culturable Bacteria from an Alpine Deciduous Forest Site.

Microbiota from Alpine forest soils are key players in carbon cycling, which can be greatly affected by climate change. The aim of this study was to evaluate the degradation potential of culturable bacterial strains isolated from an alpine deciduous forest site. Fifty-five strains were studied with regard to their phylogenetic position, growth temperature range and degradation potential for organic compounds (microtiter scale screening for lignin sulfonic acid, catechol, phenol, bisphenol A) at low (5 °C) and moderate (20 °C) temperature. Additionally, the presence of putative catabolic genes (catechol-1,2-dioxygenase, multicomponent phenol hydroxylase, protocatechuate-3,4-dioxygenase) involved in the degradation of these organic compounds was determined through PCR. The results show the importance of the Proteobacteria phylum as its representatives did show good capabilities for biodegradation and good growth at -5 °C. Overall, 82% of strains were able to use at least one of the tested organic compounds as their sole carbon source. The presence of putative catabolic genes could be shown over a broad range of strains and in relation to their degradation abilities. Subsequently performed gene sequencing indicated horizontal gene transfer for catechol-1,2-dioxygenase and protocatechuate-3,4-dioxygenase. The results show the great benefit of combining molecular and culture-based techniques.

Biodegradation of lignin monomers and bioconversion of ferulic acid to vanillic acid by Paraburkholderia aromaticivorans AR20-38 isolated from Alpine forest soil.

Lignin bio-valorization is an emerging field of applied biotechnology and has not yet been studied at low temperatures. Paraburkholderia aromaticivorans AR20-38 was examined for its potential to degrade six selected lignin monomers (syringic acid, p-coumaric acid, 4-hydroxybenzoic acid, ferulic acid, vanillic acid, benzoic acid) from different upper funneling aromatic pathways. The strain degraded four of these compounds at 10°C, 20°C, and 30°C; syringic acid and vanillic acid were not utilized as sole carbon source. The degradation of 5 mM and 10 mM ferulic acid was accompanied by the stable accumulation of high amounts of the value-added product vanillic acid (85-89% molar yield; 760 and 1540 mg l-1, respectively) over the whole temperature range tested. The presence of essential genes required for reactions in the upper funneling pathways was confirmed in the genome. This is the first report on biodegradation of lignin monomers and the stable vanillic acid production at low and moderate temperatures by P. aromaticivorans. KEY POINTS: • Paraburkholderia aromaticivorans AR20-38 successfully degrades four lignin monomers. • Successful degradation study at low (10°C) and moderate temperatures (20-30°C). • Biotechnological value: high yield of vanillic acid produced from ferulic acid.

Culturable bacteria from an Alpine coniferous forest site: biodegradation potential of organic polymers and pollutants.

The potential of the culturable bacterial community from an Alpine coniferous forest site for the degradation of organic polymers and pollutants at low (5 °C) and moderate (20 °C) temperatures was evaluated. The majority of the 68 strains belonged to the phylum Proteobacteria (77%). Other strains were related to Bacteroidetes (12%), Alphaproteobacteria (4%), Actinobacteria (3%), and Firmicutes (3%). The strains were grouped into 42 different OTUs. The highest bacterial diversity was found within the phylum Bacteroidetes. All strains, except one, could grow at temperatures from 5 to 25 °C. The production of enzyme activities involved in the degradation of organic polymers present in plant litter (carboxymethyl cellulose, microgranular cellulose, xylan, polygalacturonic acid) was almost comparable at 5 °C (68%) and 20 °C (63%). Utilizers of lignin compounds (lignosulfonic acid, lignin alkali) as sole carbon source were found to a higher extent at 20 °C (57%) than at 5 °C (24%), but the relative fractions among positively tested strains utilizing these compounds were almost identical at the two temperatures. Similar results were noted for utilizers of organic pollutants (n-hexadecane, diesel oil, phenol, glyphosate) as sole carbon source. More than two-thirds showed constitutively expressed catechol-1,2-dioxygenase activity both at 5 °C (74%) and 20 °C (66%). Complete phenol (2.5 mmol/L) degradation by strain Paraburkholderia aromaticivorans AR20-38 was demonstrated at 0-30 °C, amounts up to 7.5 mmol/L phenol were fully degraded at 10-30 °C. These results are useful to better understand the effect of changing temperatures on microorganisms involved in litter degradation and nutrient turnover in Alpine forest soils.

Draft Genome Sequence of the Bacterium Paraburkholderia aromaticivorans AR20-38, a Gram-Negative, Cold-Adapted Degrader of Aromatic Compounds.

Here, we report the draft genome sequence of Paraburkholderia aromaticivorans strain AR20-38, a cold-adapted Gram-negative bacterium. It was isolated from Alpine forest soil and can degrade a range of aromatic compounds.

Psychrophilic lifestyles: mechanisms of adaptation and biotechnological tools.

Cold-adapted microorganisms inhabiting permanently low-temperature environments were initially just a biological curiosity but have emerged as rich sources of numerous valuable tools for application in a broad spectrum of innovative technologies. To overcome the multiple challenges inherent to life in their cold habitats, these microorganisms have developed a diverse array of highly sophisticated synergistic adaptations at all levels within their cells: from cell envelope and enzyme adaptation, to cryoprotectant and chaperone production, and novel metabolic capabilities. Basic research has provided valuable insights into how these microorganisms can thrive in their challenging habitat conditions and into the mechanisms of action of the various adaptive features employed, and such insights have served as a foundation for the knowledge-based development of numerous novel biotechnological tools. In this review, we describe the current knowledge of the adaptation strategies of cold-adapted microorganisms and the biotechnological perspectives and commercial tools emerging from this knowledge. Adaptive features and, where possible, applications, in relation to membrane fatty acids, membrane pigments, the cell wall peptidoglycan layer, the lipopolysaccharide component of the outer cell membrane, compatible solutes, antifreeze and ice-nucleating proteins, extracellular polymeric substances, biosurfactants, chaperones, storage materials such as polyhydroxyalkanoates and cyanophycins and metabolic adjustments are presented and discussed.

Microbial ecology of the cryosphere (glacial and permafrost habitats): current knowledge.

Microorganisms in cold ecosystems play a key ecological role in their natural habitats. Since these ecosystems are especially sensitive to climate changes, as indicated by the worldwide retreat of glaciers and ice sheets as well as permafrost thawing, an understanding of the role and potential of microbial life in these habitats has become crucial. Emerging technologies have added significantly to our knowledge of abundance, functional activity, and lifestyles of microbial communities in cold environments. The current knowledge of microbial ecology in glacial habitats and permafrost, the most studied habitats of the cryosphere, is reported in this review.

Solimicrobium silvestre gen. nov., sp. nov., isolated from alpine forest soil.

A Gram-stain-negative, rod-shaped, motile, catalase and cytochrome c oxidase-positive bacterial strain, designated S20-91T, was isolated from alpine forest soil. Growth occurred within a temperature range of 0-25 °C. Yeast extract was required for growth. Phylogenetic analysis based on 16S rRNA gene sequencing showed that strain S20-91T was related to the genus Herminiimonas and had the highest 16S rRNA gene sequence similarity to Herminiimonas arsenicoxydans ULPAs1T (96.5 %). The strain contained ubiquinone 8 as the predominant respiratory quinone and phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol as the major polar lipids. The major cellular fatty acids (>10 %) were C16 : 1ω7c (55.3 %) and C16 : 0 (25.6 %). The genomic DNA G+C content was 47.6 mol%. Combined data of genomic, phylogenetic, phenotypic and chemotaxonomic analyses demonstrated that strain S20-91T represents a novel genus and species, for which the name Solimicrobium silvestre gen. nov., sp. nov. is proposed. The type strain is S20-91T (=DSM 104733T=LMG 30010).

Insights into microbial communities mediating the bioremediation of hydrocarbon-contaminated soil from an Alpine former military site.

The study of microbial communities involved in soil bioremediation is important to identify the specific microbial characteristics that determine improved decontamination rates. Here, we characterized bacterial, archaeal, and fungal communities in terms of (i) abundance (using quantitative PCR) and (ii) taxonomic diversity and structure (using Illumina amplicon sequencing) during the bioremediation of long-term hydrocarbon-contaminated soil from an Alpine former military site during 15 weeks comparing biostimulation (inorganic NPK fertilization) vs. natural attenuation and considering the effect of temperature (10 vs. 20 °C). Although a considerable amount of total petroleum hydrocarbon (TPH) loss could be attributed to natural attenuation, significantly higher TPH removal rates were obtained with NPK fertilization and at increased temperature, which were related to the stimulation of the activities of indigenous soil microorganisms. Changing structures of bacterial and fungal communities significantly explained shifts in TPH contents in both natural attenuation and biostimulation treatments at 10 and 20 °C. However, archaeal communities, in general, and changing abundances and diversities in bacterial and fungal communities did not play a decisive role on the effectiveness of soil bioremediation. Gammaproteobacteria and Bacteroidia classes, within bacterial community, and undescribed/novel groups, within fungal community, proved to be actively involved in TPH removal in natural attenuation and biostimulation at both temperatures.

Marmoricola silvestris sp. nov., a novel actinobacterium isolated from alpine forest soil.

A Gram-stain-positive, flagellated, catalase- and cytochrome c oxidase-positive bacterial strain, designated S20-100T, was isolated from alpine forest soil. Growth occurred at a temperature range of 0-30 °C, at pH 6-9 and in the presence of 0-1 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain S20-100T was related to the genus Marmoricola and had the highest 16S rRNA gene sequence similarity to Marmoricola ginsengisoli Gsoil 097T (98.4 %) and Marmoricola solisilvae KIS18-7T (98.3 %). The cell-wall peptidoglycan of strain S20-100T contained ll-diaminopimelic acid (ll-Dpm) as the diagnostic diamino acid and was of the type A3γ ll-Dpm - Gly. The strain contained MK-8(H4) as the predominant isoprenoid quinone and diphosphatidylglycerol, phosphatidylglycerol, four unidentified phospholipids and three unidentified lipids in lower amounts. The major cellular fatty acids (>10 %) were iso-C16 : 0, C17 : 1ω6c and C18 : 1ω9c. The genomic DNA G+C content was 66.2 mol%. Combined data of phylogenetic, phenotypic and chemotaxonomic analyses demonstrated that strain S20-100T represents a novel species of the genus Marmoricola, for which the name Marmoricola silvestris sp. nov. is proposed. The type strain is S20-100T (=DSM 104694T=LMG 30008T).

Lysobacter silvestris sp. nov., isolated from alpine forest soil, and reclassification of Luteimonas tolerans as Lysobacter tolerans comb. nov.

A Gram-stain-negative, rod-shaped, motile, catalase-positive and cytochrome c oxidase-positive bacterial strain, designated AM20-91T, was isolated from alpine forest soil. Phylogenetic analysis based on 16S rRNA gene sequencing showed that strain AM20-91T was related to the genus Lysobacter and had highest 16S rRNA gene sequence similarities to the type strains of Lysobacter novalis THG-PC7T (97.8 %), Luteimonas tolerans UM1T (97.7 %) and Lysobacter ximonensis XM415T (97.0 %). The strain contained ubiquinone 8 as the predominant respiratory quinone; its polar lipid profile contained phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and two unidentified aminophospholipids. The major cellular fatty acids (>10 %) were iso-C15 : 0, iso-C11 : 0 3-OH and iso-C11 : 0. The DNA G+C content was 63.35 % (draft genome sequence). The combined results of phylogenetic, phenotypic, DNA-DNA relatedness and chemotaxonomic analyses demonstrated that strain AM20-91T represents a novel species of the genus Lysobacter, for which the name Lysobacter silvestris sp. nov. is proposed. The type strain is AM20-91T (=DSM 104734T=LMG 30011). In this study, it is also proposed that Luteimonas tolerans be reclassified as member of the genus Lysobacter.

Characterization of soil bacterial, archaeal and fungal communities inhabiting archaeological human-impacted layers at Monte Iato settlement (Sicily, Italy).

Microbial communities in human-impacted soils of ancient settlements have been proposed to be used as ecofacts (bioindicators) of different ancient anthropogenic activities. In this study, bacterial, archaeal and fungal communities inhabiting soil of three archaic layers, excavated at the archaeological site on Monte Iato (Sicily, Italy) and believed to have been created in a chronological order in archaic times in the context of periodic cultic feasts, were investigated in terms of (i) abundance (phospholipid fatty acid (PLFA) analysis and quantitative PCR)), (ii) carbon(C)-source consumption patterns (Biolog-Ecoplates) and (iii) diversity and community composition (Illumina amplicon sequencing). PLFA analyses demonstrated the existence of living bacteria and fungi in the soil samples of all three layers. The upper layer showed increased levels of organic C, which were not concomitant with an increment in the microbial abundance. In taxonomic terms, the results indicated that bacterial, archaeal and fungal communities were highly diverse, although differences in richness or diversity among the three layers were not detected for any of the communities. However, significantly different microbial C-source utilization patterns and structures of bacterial, archaeal and fungal communities in the three layers confirmed that changing features of soil microbial communities reflect different past human activities.

Seasonal soil microbial responses are limited to changes in functionality at two Alpine forest sites differing in altitude and vegetation.

The study of soil microbial responses to environmental changes is useful to improve simulation models and mitigation strategies for climate change. We here investigated two Alpine forest sites (deciduous forest vs. coniferous forest) situated at different altitudes (altitudinal effect) in spring and autumn (seasonal effect) regarding: (i) bacterial and fungal abundances (qPCR); (ii) diversity and structure of bacterial and fungal communities (amplicon sequencing); and (iii) diversity and composition of microbial functional gene community (Geochip 5.0). Significant altitudinal changes were detected in microbial abundances as well as in diversity and composition of taxonomic and functional communities as a consequence of the differences in pH, soil organic matter (SOM) and nutrient contents and soil temperatures measured between both sites. A network analysis revealed that deciduous forest site (at lower altitude) presented a lower resistance to environmental changes than that of coniferous forest site (at higher altitude). Significant seasonal effects were detected only for the diversity (higher values in autumn) and composition of microbial functional gene community, which was related to the non-significant increased SOM and nutrient contents detected in autumn respect to spring and the presumable high capacity of soil microbial communities to respond in functional terms to discreet environmental changes.

Microbiology Meets Archaeology: Soil Microbial Communities Reveal Different Human Activities at Archaic Monte Iato (Sixth Century BC).

Microbial ecology has been recognized as useful in archaeological studies. At Archaic Monte Iato in Western Sicily, a native (indigenous) building was discovered. The objective of this study was the first examination of soil microbial communities related to this building. Soil samples were collected from archaeological layers at a ritual deposit (food waste disposal) in the main room and above the fireplace in the annex. Microbial soil characterization included abundance (cellular phospholipid fatty acids (PLFA), viable bacterial counts), activity (physiological profiles, enzyme activities of viable bacteria), diversity, and community structure (bacterial and fungal Illumina amplicon sequencing, identification of viable bacteria). PLFA-derived microbial abundance was lower in soils from the fireplace than in soils from the deposit; the opposite was observed with culturable bacteria. Microbial communities in soils from the fireplace had a higher ability to metabolize carboxylic and acetic acids, while those in soils from the deposit metabolized preferentially carbohydrates. The lower deposit layer was characterized by higher total microbial and bacterial abundance and bacterial richness and by a different carbohydrate metabolization profile compared to the upper deposit layer. Microbial community structures in the fireplace were similar and could be distinguished from those in the two deposit layers, which had different microbial communities. Our data confirmed our hypothesis that human consumption habits left traces on microbiota in the archaeological evidence; therefore, microbiological residues as part of the so-called ecofacts are, like artifacts, key indicators of consumer behavior in the past.

Psychromicrobium silvestre gen. nov., sp. nov., an actinobacterium isolated from alpine forest soils.

Two Gram-stain-variable, non-motile, catalase-positive and cytochrome c oxidase-negative bacteria, designated AK20-18T and AM20-54, were isolated from forest soil samples collected in the Italian Alps. Growth occurred at a temperature range of 5-30 °C, at pH 6-9 and in the presence of 0-5 % (w/v) NaCl. The 16S rRNA gene sequence similarity between strains AK20-18T and AM20-54 was 100 %. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain AK20-18T had highest 16S rRNA gene sequence similarity with the type strain of Arthrobacter psychrochitiniphilus (96.9 %). The cell-wall peptidoglycan structure of strain AK20-18T was of the type A3alpha l-Lys-l-Thr-l-Ala2 (A11.27). The whole-cell sugars were galactose, ribose and lesser amounts of mannose. The major respiratory quinone of the two strains was menaquinone 9(H2) [MK-9(H2)], whereas MK-10(H2) was a minor component. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and unknown glycolipids. The major cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The genomic DNA G+C content was 59.9 mol%. Combined data of phylogenetic, phenotypic and chemotaxonomic analyses demonstrated that strains AK20-18T and AM20-54 represent a novel genus and species, for which the name Psychromicrobium silvestre gen. nov., sp. nov. is proposed. The type strain of Psychromicrobium silvestregen. nov., sp. nov. is AK20-18T (=DSM 102047T=LMG 29369T).

Nakamurella silvestris sp. nov., an actinobacterium isolated from alpine forest soil.

A Gram-stain-positive, non-motile, catalase-positive and cytochrome c oxidase-negative bacterium, designated strain S20-107T, was isolated from alpine forest soil. Growth occurred at 0-30 °C, at pH 6-9 and in the presence of 0-3 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain S20-107T was related to the genus Nakamurella and had the highest 16S rRNA gene sequence similarity with Nakamurella flavida DS-52T (96.1 %). Strain S20-107T showed <96.1% 16S rRNA gene sequence similarities to all other recognized members of the genus Nakamurella. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The major whole-cell sugars were glucose, galactose, mannose, arabinose, ribose and rhamnose. The strain contained MK-8(H4) as the predominant menaquinone and diphosphatidylglycerol, phosphatidylethanolamine and an unidentified aminophospholipid as the major polar lipids. The major cellular fatty acids were anteiso-C15 : 0, C16 : 0, summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c and/or iso-C15 : 0 2-OH) and iso-C16 : 0. The genomic DNA G+C content was 70.5 mol%. Combined data of phylogenetic, phenotypic and chemotaxonomic analyses demonstrated that strain S20-107T represents a novel species of the genus Nakamurella, for which the name Nakamurella silvestris sp. nov. is proposed. The type strain is S20-107T (=DSM 102309T=LMG 29427T).

Seasonal and altitudinal changes of culturable bacterial and yeast diversity in Alpine forest soils.

The effect of altitude and season on abundance and diversity of the culturable heterotrophic bacterial and yeast community was examined at four forest sites in the Italian Alps along an altitude gradient (545-2000 m). Independently of altitude, bacteria isolated at 0 °C (psychrophiles) were less numerous than those recovered at 20 °C. In autumn, psychrophilic bacterial population increased with altitude. The 1194 bacterial strains were primarily affiliated with the classes Alpha-, Beta-, Gammaproteobacteria, Spingobacteriia and Flavobacteriia. Fifty-seven of 112 operational taxonomic units represented potential novel species. Strains isolated at 20 °C had a higher diversity and showed similarities in taxa composition and abundance, regardless of altitude or season, while strains isolated at 0 °C showed differences in community composition at lower and higher altitudes. In contrast to bacteria, yeast diversity was season-dependent: site- and altitude-specific effects on yeast diversity were only detected in spring. Isolation temperature affected the relative proportions of yeast genera. Isolations recovered 719 strains, belonging to the classes Dothideomycetes, Saccharomycetes, Tremellomycetes and Mycrobotryomycetes. The presence of few dominant bacterial OTUs and yeast species indicated a resilient microbial population that is not affected by season or altitude. Soil nutrient contents influenced significantly abundance and diversity of culturable bacteria, but not of culturable yeasts.