Enzymatic machinery of wood-inhabiting fungi that degrade temperate tree species.

Deadwood provides habitat for fungi and serves diverse ecological functions in forests. We already have profound knowledge of fungal assembly processes, physiological and enzymatic activities, and resulting physico-chemical changes during deadwood decay. However, in situ detection and identification methods, fungal origins, and a mechanistic understanding of the main lignocellulolytic enzymes are lacking. This study used metaproteomics to detect the main extracellular lignocellulolytic enzymes in 12 tree species in a temperate forest that have decomposed for 8 ½ years. Mainly white-rot (and few brown-rot) Basidiomycota were identified as the main wood decomposers, with Armillaria as the dominant genus; additionally, several soft-rot xylariaceous Ascomycota were identified. The key enzymes involved in lignocellulolysis included manganese peroxidase, peroxide-producing alcohol oxidases, laccase, diverse glycoside hydrolases (cellulase, glucosidase, xylanase), esterases, and lytic polysaccharide monooxygenases. The fungal community and enzyme composition differed among the 12 tree species. Ascomycota species were more prevalent in angiosperm logs than in gymnosperm logs. Regarding lignocellulolysis as a function, the extracellular enzyme toolbox acted simultaneously and was interrelated (e.g. peroxidases and peroxide-producing enzymes were strongly correlated), highly functionally redundant, and present in all logs. In summary, our in situ study provides comprehensive and detailed insight into the enzymatic machinery of wood-inhabiting fungi in temperate tree species. These findings will allow us to relate changes in environmental factors to lignocellulolysis as an ecosystem function in the future.

Active soil microbial composition and proliferation are directly affected by the presence of biocides from building materials.

Combinations of biocides are commonly added to building materials to prevent microbial growth and thereby cause degradation of the façades. These biocides reach the environment by leaching from façades posing an environmental risk. Although ecotoxicity to the aquatic habitat is well established, there is hardly any data on the ecotoxicological effects of biocides on the soil habitat. This study aimed to characterize the effect of the biocides terbutryn, isoproturon, octhilinone, and combinations thereof on the total and metabolically active soil microbial community composition and functions. Total soil microbial community was retrieved directly from the nucleic acid extracts, while the DNA of the active soil microbial community was separated after bromodeoxyuridine labeling. Bacterial 16S rRNA gene and fungal internal transcribed spacer region gene-based amplicon sequencing was carried out for both active and total, while gene copy numbers were quantified only for the total soil microbial community. Additionally, soil respiration and physico-chemical parameters were analyzed to investigate overall soil microbial activity. The bacterial and fungal gene copy numbers were significantly affected by single biocides and combined biocide soil treatment but not soil respiration and physico-chemical parameters. While the total soil microbiome experienced only minor effects from single and combined biocide treatment, the active soil microbiome was significantly impacted in its diversity, richness, composition, and functional patterns. The active bacterial richness was more sensitive than fungal richness. However, the adverse effects of the biocide combination treatments on soil bacterial richness were highly dependent on the identities of the biocide combination. Our results demonstrate that the presence of biocides frequently used in building materials affects the active soil microbiome. Thereby, the approach described herein can be used as an ecotoxicological measure for the effect on complex soil environments in future studies.

Biological nitrogen fixation, diversity and community structure of diazotrophs in two mosses in 25 temperate forests.

Many moss species are associated with nitrogen (N)-fixing bacteria (diazotrophs) that support the N supply of mosses. Our knowledge relates primarily to pristine ecosystems with low atmospheric N input, but knowledge of biological N fixation (BNF) and diazotrophic communities in mosses in temperate forests with high N deposition is limited. We measured BNF rates using the direct stable isotope method and studied the total and potentially active diazotrophic communities in two abundant mosses, Brachythecium rutabulum and Hypnum cupressiforme, both growing on lying deadwood trunks in 25 temperate forest sites. BNF rates in both mosses were similar to those observed in moss species of pristine ecosystems. H. cupressiforme fixed three times more N2 and exhibited lower diazotrophic richness than B. rutabulum. Frankia was the most prominent diazotroph followed by cyanobacteria Nostoc. Manganese, iron, and molybdenum contents in mosses were positively correlated with BNF and diazotrophic communities. Frankia maintained high BNF rates in H. cupressiforme and B. rutabulum even under high chronic N deposition in Central European forests. Moss N concentration and 15 N abundance indicate a rather minor contribution of BNF to the N nutrition of these mosses.

Tree mycorrhizal type regulates leaf and needle microbial communities, affects microbial assembly and co-occurrence network patterns, and influences litter decomposition rates in temperate forest.

Background: Tree mycorrhizal types (arbuscular mycorrhizal fungi and ectomycorrhizal fungi) alter nutrient use traits and leaf physicochemical properties and, thus, affect leaf litter decomposition. However, little is known about how different tree mycorrhizal species affect the microbial diversity, community composition, function, and community assembly processes that govern leaf litter-dwelling microbes during leaf litter decomposition. Methods: In this study, we investigated the microbial diversity, community dynamics, and community assembly processes of nine temperate tree species using high-resolution molecular technique (Illumina sequencing), including broadleaved arbuscular mycorrhizal, broadleaved ectomycorrhizal, and coniferous ectomycorrhizal tree types, during leaf litter decomposition. Results and discussion: The leaves and needles of different tree mycorrhizal types significantly affected the microbial richness and community composition during leaf litter decomposition. Leaf litter mass loss was related to higher sequence reads of a few bacterial functional groups, particularly N-fixing bacteria. Furthermore, a link between bacterial and fungal community composition and hydrolytic and/or oxidative enzyme activity was found. The microbial communities in the leaf litter of different tree mycorrhizal types were governed by different proportions of determinism and stochasticity, which changed throughout litter decomposition. Specifically, determinism (mainly variable selection) controlling bacterial community composition increased over time. In contrast, stochasticity (mainly ecological drift) increasingly governed fungal community composition. Finally, the co-occurrence network analysis showed greater competition between bacteria and fungi in the early stages of litter decomposition and revealed a contrasting pattern between mycorrhizal types. Conclusion: Overall, we conclude that tree mycorrhizal types influence leaf litter quality, which affects microbial richness and community composition, and thus, leaf litter decomposition.

Fungicides and insecticides can alter the microbial community on the cuticle of honey bees.

Honey bees are crucial for our ecosystems as pollinators, but the intensive use of plant protection products (PPPs) in agriculture poses a risk for them. PPPs do not only affect target organisms but also affect non-targets, such as the honey bee Apis mellifera and their microbiome. This study is the first of its kind, aiming to characterize the effect of PPPs on the microbiome of the cuticle of honey bees. We chose PPPs, which have frequently been detected in bee bread, and studied their effects on the cuticular microbial community and function of the bees. The effects of the fungicide Difcor® (difenoconazole), the insecticide Steward® (indoxacarb), the combination of both (mix A) and the fungicide Cantus® Gold (boscalid and dimoxystrobin), the insecticide Mospilan® (acetamiprid), and the combination of both (mix B) were tested. Bacterial 16S rRNA gene and fungal transcribed spacer region gene-based amplicon sequencing and quantification of gene copy numbers were carried out after nucleic acid extraction from the cuticle of honey bees. The treatment with Steward® significantly affected fungal community composition and function. The fungal gene copy numbers were lower on the cuticle of bees treated with Difcor®, Steward®, and PPP mix A in comparison with the controls. However, bacterial and fungal gene copy numbers were increased in bees treated with Cantus® Gold, Mospilan®, or PPP mix B compared to the controls. The bacterial cuticular community composition of bees treated with Cantus® Gold, Mospilan®, and PPP mix B differed significantly from the control. In addition, Mospilan® on its own significantly changed the bacterial functional community composition. Cantus® Gold significantly affected fungal gene copy numbers, community, and functional composition. Our results demonstrate that PPPs show adverse effects on the cuticular microbiome of honey bees and suggest that PPP mixtures can cause stronger effects on the cuticular community than a PPP alone. The cuticular community composition was more diverse after the PPP mix treatments. This may have far-reaching consequences for the health of honey bees.

Solitary Bees Host More Bacteria and Fungi on Their Cuticle than Social Bees.

Bees come into contact with bacteria and fungi from flowering plants during their foraging trips. The Western honeybee (Apis mellifera) shows a pronounced hygienic behavior with social interactions, while the solitary red mason bee (Osmia bicornis) lacks a social immune system. Since both visit the same floral resources, it is intriguing to speculate that the body surface of a solitary bee should harbor a more complex microbiome than that of the social honeybee. We compared the cuticular microbiomes of A. mellifera (including three European subspecies) and O. bicornis for the first time by bacterial 16S rRNA and fungal ITS gene-based high-throughput amplicon sequencing. The cuticular microbiome of the solitary O. bicornis was significantly more complex than that of the social A. mellifera. The microbiome composition of A. mellifera subspecies was very similar. However, we counted significantly different numbers of fungi and a higher diversity in the honeybee subspecies adapted to warmer climates. Our results suggest that the cuticular microbiome of bees is strongly affected by visited plants, lifestyle and adaptation to temperature, which have important implications for the maintenance of the health of bees under conditions of global change.

Nitrogen addition increases mass loss of gymnosperm but not of angiosperm deadwood without changing microbial communities.

Enhanced nitrogen (N) deposition due to combustion of fossil fuels and agricultural fertilization is a global phenomenon which has severely altered carbon (C) and N cycling in temperate forest ecosystems in the northern hemisphere. Although deadwood holds a substantial amount of C in forest ecosystems and thus plays a crucial role in nutrient cycling, the effect of increased N deposition on microbial processes and communities, wood chemical traits and deadwood mass loss remains unclear. Here, we simulated high N deposition rates by adding reactive N in form of ammonium-nitrate (40 kg N ha-1 yr-1) to deadwood of 13 temperate tree species over nine years in a field experiment in Germany. Non-treated deadwood from the same logs served as control with background N deposition. Our results show that chronically elevated N levels alters deadwood mass loss alongside respiration, enzymatic activities and wood chemistry depending on tree clade and species. In gymnosperm deadwood, elevated N increased mass loss by +38 %, respiration by +37 % and increased laccase activity 12-fold alongside increases of white-rot fungal abundance +89 % (p = 0.03). Furthermore, we observed marginally significant (p = 0.06) shifts of bacterial communities in gymnosperm deadwood. In angiosperm deadwood, we did not detect consistent effects on mass loss, physico-chemical properties, extracellular enzymatic activity or changes in microbial communities except for changes in abundance of 10 fungal OTUs in seven tree species and 28 bacterial OTUs in 10 tree species. We conclude that N deposition alters decomposition processes exclusively in N limited gymnosperm deadwood in the long term by enhancing fungal activity as expressed by increases in respiration rate and extracellular enzyme activity with minor shifts in decomposing microbial communities. By contrast, deadwood of angiosperm tree species had higher N concentrations and mass loss as well as community composition did not respond to N addition.

Learning Needle Placement in Soft Tissue With Robot-assisted Navigation.

BACKGROUND/AIM: The aim of this phantom study was to evaluate the learning curves of novices practicing how to place a cone-beam computed tomography (CBCT)-guided needle using a novel robotic assistance system (RAS). MATERIALS AND METHODS: Ten participants performed 18 punctures each with random trajectories in a phantom setting, supported by a RAS over 3 days. Precision, duration of the total intervention, duration of the needle placement, autonomy, and confidence of the participants were measured, displaying possible learning curves. RESULTS: No statistically significant differences were observed in terms of needle tip deviation during the trial days (mean deviation day 1: 2.82 mm; day 3: 3.07 mm; p=0.7056). During the trial days, the duration of the total intervention (mean duration: day 1: 11:22 min; day 3: 07:39 min; p<0.0001) and the duration of the needle placement decreased (mean duration: day 1: 03:17 min; day 3: 02:11 min; p<0.0001). In addition, autonomy (mean percentage of achievable points: day 1: 94%; day 3: 99%; p<0.0001) and confidence of the participants (mean percentage of achievable points: day 1: 78%; day 3: 91%; p<0.0001) increased significantly during the trial days. CONCLUSION: The participants were already able to carry out the intervention precisely using the RAS on the first day of the trial. Throughout the trial, the participants' performance improved in terms of duration and confidence.

Fate of a biodegradable plastic in forest soil: Dominant tree species and forest types drive changes in microbial community assembly, influence the composition of plastisphere, and affect poly(butylene succinate-co-adipate) degradation.

Poly(butylene succinate-co-adipate) (PBSA) degradation and its plastisphere microbiome in cropland soils have been studied; however, such knowledge is limited in the case of forest ecosystems. In this context, we investigated: i) the impact of forest types (conifer and broadleaved forests) on the plastisphere microbiome and its community assembly, ii) their link to PBSA degradation, and iii) the identities of potential microbial keystone taxa. We determined that forest type significantly affected microbial richness (F = 5.26-9.88, P = 0.034 to 0.006) and fungal community composition (R2 = 0.38, P = 0.001) of the plastisphere microbiome, whereas its effects on microbial abundance and bacterial community composition were not significant. The bacterial community was governed by stochastic processes (mainly homogenizing dispersal), whereas the fungal community was driven by both stochastic and deterministic processes (drift and homogeneous selection). The highest molar mass loss was found for PBSA degraded under Pinus sylvestris (26.6 ± 2.6 to 33.9 ± 1.8 % (mean ± SE) at 200 and 400 days, respectively), and the lowest molar mass loss was found under Picea abies (12.0 ± 1.6 to 16.0 ± 0.5 % (mean ± SE) at 200 and 400 days, respectively). Important fungal PBSA decomposers (Tetracladium) and atmospheric dinitrogen (N2)-fixing bacteria (symbiotic: Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium and Methylobacterium and non-symbiotic: Mycobacterium) were identified as potential keystone taxa. The present study is among the first to determine the plastisphere microbiome and its community assembly processes associated with PBSA in forest ecosystems. We detected consistent biological patterns in the forest and cropland ecosystems, indicating a potential mechanistic interaction between N2-fixing bacteria and Tetracladium during PBSA biodegradation.

Co-Infection of Potential Tick-Borne Pathogens of the Order Rickettsiales and Borrelia burgdorferi s. l. and Their Link to Season and Area in Germany.

The prevalence of potential human pathogenic members of the order Rickettsiales differs between Borrelia burgdorferi sensu lato-positive and -negative tick microbiomes. Here, co-infection of members of the order Rickettsiales, such as Rickettsia spp., Anaplasma phagocytophilum, Wolbachia pipientis, and Neoehrlichia mikurensis as well as B. burgdorferi s.l. in the tick microbiome was addressed. This study used conventional PCRs to investigate the diversity and prevalence of the before-mentioned bacteria in 760 nucleic acid extracts of I. ricinus ticks detached from humans, which were previously tested for B. burgdorferi s.l.. A gltA gene-based amplicon sequencing approach was performed to identify Rickettsia species. The prevalence of Rickettsia spp. (16.7%, n = 127) and W. pipientis (15.9%, n = 121) were similar, while A. phagocytophilum was found in 2.8% (n = 21) and N. mikurensis in 0.1% (n = 1) of all ticks. Co-infection of B. burgdorferi s. l. with Rickettsia spp. was most frequent. The gltA gene sequencing indicated that Rickettsia helvetica was the dominant Rickettsia species in tick microbiomes. Moreover, R, monacensis and R. raoultii were correlated with autumn and area south, respectively, and a negative B. burgdorferi s. l. finding. Almost every fifth tick carried DNA of at least two of the human pathogenic bacteria studied here.

FungalTraits vs. FUNGuild: Comparison of Ecological Functional Assignments of Leaf- and Needle-Associated Fungi Across 12 Temperate Tree Species.

Recently, a new annotation tool "FungalTraits" was created based on the previous FUNGuild and FunFun databases, which has attracted high attention in the scientific community. These databases were widely used to gain more information from fungal sequencing datasets by assigning fungal functional traits. More than 1500 publications so far employed FUNGuild and the aim of this study is to compare this successful database with the recent FungalTraits database. Quality and quantity of the assignment by FUNGuild and FungalTraits to a fungal internal transcribed spacer (ITS)-based amplicon sequencing dataset on amplicon sequence variants (ASVs) were addressed. Sequencing dataset was derived from leaves and needles of 12 temperate broadleaved and coniferous tree species. We found that FungalTraits assigned more functional traits than FUNGuild, and especially the coverage of saprotrophs, plant pathogens, and endophytes was higher while lichenized fungi revealed similar findings. Moreover, ASVs derived from leaves and needles of each tree species were better assigned to all available fungal traits as well as to saprotrophs by FungalTraits compared to FUNGuild in particular for broadleaved tree species. Assigned ASV richness as well as fungal functional community composition was higher and more diverse after analyses with FungalTraits compared to FUNGuild. Moreover, datasets of both databases showed similar effect of environmental factors for saprotrophs but for endophytes, unidentical patterns of significant corresponding factors were obtained. As a conclusion, FungalTraits is superior to FUNGuild in assigning a higher quantity and quality of ASVs as well as a higher frequency of significant correlations with environmental factors.

A poisoned apple: First insights into community assembly and networks of the fungal pathobiome of healthy-looking senescing leaves of temperate trees in mixed forest ecosystem.

Despite the abundance of observations of foliar pathogens, our knowledge is severely lacking regarding how the potential fungal pathobiome is structured and which processes determine community assembly. In this study, we addressed these questions by analysing the potential fungal pathobiome associated with the senescing leaves and needles of 12 temperate tree species. We compared fungal plant pathogen load in the senescing leaves/needles and demonstrated that healthy-looking leaves/needles are inhabited by diverse and distinct fungal plant pathogens. We detected 400 fungal plant pathogenic ASVs belonging to 130 genera. The fungal plant pathogenic generalist, Mycosphaerella, was found to be the potential most significant contributor to foliar disease in seedlings. The analyses of assembly process and co-occurrence network showed that the fungal plant pathogenic communities in different tree types are mainly determined by stochastic processes. However, the homogenising dispersal highly contributes in broadleaf trees, whereas ecological drift plays an important role in coniferious trees. The deterministic assembly processes (dominated by variable selection) contributed more in broadleaf trees as compared to coniferous trees. We found that pH and P level significantly corresponded with fungal plant pathogenic community compositions in both tree types. Our study provides the first insight and mechanistic understanding into the community assembly, networks, and complete taxonomy of the foliar fungal pathobiome in senescing leaves and needles.

Dark side of a bio-based and biodegradable plastic? Assessment of pathogenic microbes associated with poly(butylene succinate-co-adipate) under ambient and future climates using next-generation sequencing.

Bio-based and biodegradable plastic mulching films have been proposed to replace the non-biodegradable plastic mulch films to solve plastic pollution problems in agricultural soils. However, the impact of bio-based and biodegradable plastics on plant and human health remains largely unexplored. Here, we aimed to assess the risk under field conditions of a bio-based and biodegradable poly(butylene succinate-co-adipate; PBSA), a widely used mulching film as carrier of potential pathogenic microorganisms (bacteria and fungi) at ambient and future climate conditions. Overall, we affiliated 64 fungal and 11 bacterial operational taxonomic units (OTUs) as pathogens by using Next-Generation Sequencing approach. Our results revealed that PBSA hosted at least 53 plant pathogens, of which 51 were classified as fungi, while the other two were bacteria. Most fungal plant pathogens were able to withstand the anticipated future climate changes. We detected 13 fungal and eight bacterial OTUs, which were classified as opportunistic human pathogens. Only one bacterial OTU (Enterococcus faecium) was assigned to a human pathogen. While future climate conditions only significantly impacted on the presence and frequency of detection of few pathogens, incubation time was found to significantly impacted on nine pathogens. This result demonstrates the temporal dynamics of pathogens associated with PBSA. The threats to plant and human health were discussed. We emphasize that the risks to human health are relatively low because we mainly found opportunistic pathogens associated with PBSA and the amount are comparable to the plant debris. However, the risks to plant health may be considered as moderate because many plant pathogens were discovered and/or enriched in PBSA. Furthermore, in soil environments, the pathogenic risk of plastic is highly depending on the surrounding soil pathobiome where plastic is being decomposed.

More than you can see: Unraveling the ecology and biodiversity of lichenized fungi associated with leaves and needles of 12 temperate tree species using high-throughput sequencing.

Currently, lichen surveys are generally based on the examination of fruiting bodies. Lichens in the mycelial stage, in spores, or awaiting conditions for fruiting body formation are usually overlooked, even though they are important for maintaining biodiversity and ecosystem functions. This study aimed to explore the lichenized fungal community composition and richness associated with leaves and needles of 12 temperate tree species using Illumina MiSeq-based amplicon sequencing of the internal transcribed spacer (ITS) 2 region. Picea abies harbored the highest richness and number of lichenized fungal species. We found that the lichenized fungus Physcia adscendens dominated the leaves and needles of the most temperate tree species. Eleven lichenized fungal species detected in this study were recorded for the first time on leaves and needles. In addition, we identified Athallia cerinella, Fellhanera bouteillei, and Melanohalea exasperata that are on the German national red lists. Lichenized fungal richness was higher in conifer compared to broadleaf trees. Overall, tree species (within coniferous trees) and tree types (broadleaved vs. coniferous trees) harbored significantly different lichenized fungal community compositions pointing out the importance of host species. Diversity and community composition patterns of lichenized fungi were correlated mainly with tree species. Our study demonstrates that the diversity of foliicolous lichens associated with leaves and needles of 12 temperate tree species can be appropriately analyzed and functionally assigned using the ITS-based high-throughput sequencing. We highlighted the importance of conifers for maintaining the biodiversity of foliicolous lichens. Based on the discovery of many red list lichens, our methodological approach and results are important contributions to subsequent actions in the bio-conversation approaches.

Associated bacterial microbiome responds opportunistic once algal host Scenedesmus vacuolatus is attacked by endoparasite Amoeboaphelidium protococcarum.

The interactions of microalgae and their associated microbiomes have come to the fore of applied phycological research in recent years. However, the functional mechanisms of microalgal interactions remain largely unknown. Here, we examine functional protein patterns of the microalgae Scenedesmus vacuolatus and its associated bacterial community during algal infection by the endoparasite Amoeboaphelidium protococcarum. We performed metaproteomics analyses of non-infected (NI) and aphelid-infected (AI) S. vacuolatus cultures to investigate underlying functional and physiological changes under infectious conditions. We observed an increase in bacterial protein abundance as well as a severe shift of bacterial functional patterns throughout aphelid-infection in comparison to NI treatment. Most of the bacterial proteins (about 55%) upregulated in AI were linked to metabolism and transport of amino acids, lipids, coenzymes, nucleotides and carbohydrates and to energy production. Several proteins associated with pathogenic bacterial-plant interactions showed higher protein abundance levels in AI treatment. These functional shifts indicate that associated bacteria involved in commensalistic or mutualistic interactions in NI switch to opportunistic lifestyles and facilitate pathogenic or saprotrophic traits in AI treatment. In summary, the native bacterial microbiome adapted its metabolism to algal host die off and is able to metabolize nutrients from injured cells or decompose dead cellular material.

Links among Microbial Communities, Soil Properties and Functions: Are Fungi the Sole Players in Decomposition of Bio-Based and Biodegradable Plastic?

The incomplete degradation of bio-based and biodegradable plastics (BBPs) in soils causes multiple threats to soil quality, human health, and food security. Plastic residuals can interact with soil microbial communities. We aimed to link the structure and enzyme-mediated functional traits of a microbial community composition that were present during poly (butylene succinate-co-butylene adipate (PBSA) decomposition in soil with (PSN) and without (PS) the addition of nitrogen fertilizer ((NH4)2SO4). We identified bacterial (Achromobacter, Luteimonas, Rhodanobacter, and Lysobacter) and fungal (Fusarium, Chaetomium, Clonostachys, Fusicolla, and Acremonium) taxa that were linked to the activities of ß-glucosidase, chitinase, phosphatase, and lipase in plastic-amended soils. Fungal biomass increased by 1.7 and 4 times in PS and PSN treatment, respectively, as compared to non-plastic amended soil. PBSA significantly changed the relationships between soil properties (C: N ratio, TN, and pH) and microbial community structure; however, the relationships between fungal biomass and soil enzyme activities remained constant. PBSA significantly altered the relationship between fungal biomass and acid phosphatase. We demonstrated that although the soil functions related to nutrient cycling were not negatively affected in PSN treatment, potential negative effects are reasoned by the enrichment of plant pathogens. We concluded that in comparison to fungi, the bacteria demonstrated a broader functional spectrum in the BBP degradation process.

Cross-kingdom interactions and functional patterns of active microbiota matter in governing deadwood decay.

Microbial community members are the primary microbial colonizers and active decomposers of deadwood. This study placed sterilized standardized beech and spruce sapwood specimens on the forest ground of 8 beech- and 8 spruce-dominated forest sites. After 370 days, specimens were assessed for mass loss, nitrogen (N) content and 15N isotopic signature, hydrolytic and lignin-modifying enzyme activities. Each specimen was incubated with bromodeoxyuridine (BrdU) to label metabolically active fungal and bacterial community members, which were assessed using amplicon sequencing. Fungal saprotrophs colonized the deadwood accompanied by a distinct bacterial community that was capable of cellulose degradation, aromatic depolymerization, and N2 fixation. The latter were governed by the genus Sphingomonas, which was co-present with the majority of saprotrophic fungi regardless of whether beech or spruce specimens were decayed. Moreover, the richness of the diazotrophic Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium group was significantly correlated with mass loss, N content and 15N isotopic signature. By contrast, presence of obligate predator Bdellovibrio spp. shifted bacterial community composition and were linked to decreased beech deadwood decay rates. Our study provides the first account of the composition and function of metabolically active wood-colonizing bacterial and fungal communities, highlighting cross-kingdom interactions during the early and intermediate stages of wood decay.

Leptolyngbya sp. NIVA-CYA 255, a Promising Candidate for Poly(3-hydroxybutyrate) Production under Mixotrophic Deficiency Conditions.

Cyanobacteria are a promising source for the sustainable production of biodegradable bioplastics such as poly(3-hydroxybutyrate) (PHB). The auto-phototrophic biomass formation is based on light and CO2, which is an advantage compared to heterotrophic PHB-producing systems. So far, only a handful of cyanobacterial species suitable for the high-yield synthesis of PHB have been reported. In the present study, the PHB formation, biomass, and elemental composition of Leptolyngbya sp. NIVA-CYA 255 were investigated. Therefore, a three-stage cultivation process was applied, consisting of a growth stage; an N-, P-, and NP-depleted phototrophic stage; and a subsequent mixotrophic deficiency stage, initiated by sodium acetate supplementation. The extracted cyanobacterial PHB was confirmed by FTIR- and GC-MS analyses. Furthermore, the fluorescent dyes LipidGreen2 and Nile red were used for fluorescence-based monitoring and the visualization of PHB. LipidGreen2 was well suited for PHB quantification, while the application of Nile red was limited by fluorescence emission crosstalk with phycocyanin. The highest PHB yields were detected in NP- (325 mg g-1) and N-deficiency (213 mg g-1). The glycogen pool was reduced in all cultures during mixotrophy, while lipid composition was not affected. The highest glycogen yield was formed under N-deficiency (217 mg g-1). Due to the high carbon storage capacity and PHB formation, Leptolyngbya sp. NIVA-CYA 255 is a promising candidate for PHB production. Further work will focus on upscaling to a technical scale and monitoring the formation by LipidGreen2-based fluorometry.

In situ quantification of poly(3-hydroxybutyrate) and biomass in Cupriavidus necator by a fluorescence spectroscopic assay.

Fluorescence spectroscopy offers a cheap, simple, and fast approach to monitor poly(3-hydroxybutyrate) (PHB) formation, a biodegradable polymer belonging to the biodegradable polyester class polyhydroxyalkanoates. In the present study, a fluorescence and side scatter-based spectroscopic setup was developed to monitor in situ biomass, and PHB formation of biotechnological applied Cupriavidus necator strain. To establish PHB quantification of C. necator, the dyes 2,2-difluoro-4,6,8,10,12-pentamethyl-3-aza-1-azonia-2-boranuidatricyclo[7.3.0.03,7]dodeca-1(12),4,6,8,10-pentaene (BODIPY493/503), ethyl 5-methoxy-1,2-bis(3-methylbut-2-enyl)-3-oxoindole-2-carboxylate (LipidGreen2), and 9-(diethylamino)benzo[a]phenoxazin-5-one (Nile red) were compared with each other. Fluorescence staining efficacy was obtained through 3D-excitation-emission matrix and design of experiments. The coefficients of determination were ≥ 0.98 for all three dyes and linear to the high-pressure liquid chromatography obtained PHB content, and the side scatter to the biomass concentration. The fluorescence correlation models were further improved by the incorporation of the biomass-related side scatter. Afterward, the resulting regression fluorescence models were successfully applied to nitrogen-deficit, phosphor-deficit, and NaCl-stressed C. necator cultures. The highest transferability of the regression models was shown by using LipidGreen2. The novel approach opens a tailor-made way for a fast and simultaneous detection of the crucial biotechnological parameters biomass and PHB content during fermentation. KEY POINTS: • Intracellular quantification of PHB and biomass using fluorescence spectroscopy. • Optimizing fluorescence staining conditions and 3D-excitation-emission matrix. • PHB was best obtained by LipidGreen2, followed by BODIPDY493/503 and Nile red.

Nitrogen fixing bacteria facilitate microbial biodegradation of a bio-based and biodegradable plastic in soils under ambient and future climatic conditions.

We discovered a biological mechanism supporting microbial degradation of bio-based poly(butylene succinate-co-adipate) (PBSA) plastic in soils under ambient and future climates. Here, we show that nitrogen-fixing bacteria facilitate the microbial degradation of PBSA by enhancing fungal abundance, accelerating plastic-degrading enzyme activities, and shaping/interacting with plastic-degrading fungal communities.