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Biodegradation of polyester polyurethane by the marine fungus Cladosporium halotolerans 6UPA1
(2022)
Lack of degradability and the accumulation of polymeric wastes increase the risk for the health of the environment. Recently, recycling of polymeric waste materials becomes increasingly important as raw materials for polymer synthesis are in short supply due to the rise in price and supply chain disruptions. As an important polymer, polyurethane (PU) is widely used in modern life, therefore, PU biodegradation is desirable to avoid its accumulation in the environment. In this study, we isolated a fungal strain Cladosporium halotolerans from the deep sea which can grow in mineral medium with a polyester PU (Impranil DLN) as a sole carbon source. Further, we demonstrate that it can degrade up to 80% of Impranil PU after 3 days of incubation at 28 celcius by breaking the carbonyl groups (1732 cm(-1)) and C-N-H bonds (1532 cm(-1) and 1247 cm(-1)) as confirmed by Fourier-transform infrared (FTIR) spectroscopy analysis. Gas chromatography-mass spectrometry (GC-MS) analysis revealed polyols and alkanes as PU degradation intermediates, indicating the hydrolysis of ester and urethane bonds. Esterase and urease activities were detected in 7 days-old cultures with PU as a carbon source. Transcriptome analysis showed a number of extracellular protein genes coding for enzymes such as cutinase, lipase, peroxidase and hydrophobic surface binding proteins A (HsbA) were expressed when cultivated on Impranil PU. The yeast two-hybrid assay revealed that the hydrophobic surface binding protein ChHsbA1 directly interacts with inducible esterases, ChLip1 (lipase) and ChCut1 (cutinase). Further, the KEGG pathway for "fatty acid degradation " was significantly enriched in Impranil PU inducible genes, indicating that the fungus may use the degradation intermediates to generate energy via this pathway. Taken together, our data indicates secretion of both esterase and hydrophobic surface binding proteins by C. halotolerans plays an important role in Impranil PU absorption and subsequent degradation. Our study provides a mechanistic insight into Impranil PU biodegradation by deep sea fungi and provides the basis for future development of biotechnological PU recycling.
Biogenic greenhouse gas emissions, e.g., of methane (CH4) and carbon dioxide (CO2) from inland waters, contribute substantially to global warming. In aquatic systems, dissolved greenhouse gases are highly heterogeneous in both space and time. To better understand the biological and physical processes that affect sources and sinks of both CH4 and CO2, their dissolved concentrations need to be measured with high spatial and temporal resolution. To achieve this goal, we developed the Fast-Response Automated Gas Equilibrator (FaRAGE) for real-time in situ measurement of dissolved CH4 and CO2 concentrations at the water surface and in the water column. FaRAGE can achieve an exceptionally short response time (t(95%) = 12 s when including the response time of the gas analyzer) while retaining an equilibration ratio of 62.6% and a measurement accuracy of 0.5% for CH4. A similar performance was observed for dissolved CO2 (t(95%) = 10 s, equilibration ratio 67.1 %). An equilibration ratio as high as 91.8% can be reached at the cost of a slightly increased response time (16 s). The FaRAGE is capable of continuously measuring dissolved CO2 and CH4 concentrations in the nM-to-submM (10(-9)-10(-3) mol L-1) range with a detection limit of subnM (10(-10) mol L-1), when coupling with a cavity ring-down greenhouse gas analyzer (Picarro GasScouter). FaRAGE allows for the possibility of mapping dissolved concentration in a "quasi" three-dimensional manner in lakes and provides an inexpensive alternative to other commercial gas equilibrators. It is simple to operate and suitable for continuous monitoring with a strong tolerance for suspended particles. While the FaRAGE is developed for inland waters, it can be also applied to ocean waters by tuning the gas-water mixing ratio. The FaRAGE is easily adapted to suit other gas analyzers expanding the range of potential applications, including nitrous oxide and isotopic composition of the gases.
Freshwater fungi are a poorly studied ecological group that includes a high taxonomic diversity. Most studies on aquatic fungal diversity have focused on single habitats, thus the linkage between habitat heterogeneity and fungal diversity remains largely unexplored. We took 216 samples from 54 locations representing eight different habitats in the meso-oligotrophic, temperate Lake Stechlin in North-East Germany. These included the pelagic and littoral water column, sediments, and biotic substrates. We performed high throughput sequencing using the Roche 454 platform, employing a universal eukaryotic marker region within the large ribosomal subunit (LSU) to compare fungal diversity, community structure, and species turnover among habitats. Our analysis recovered 1027 fungal OTUs (97% sequence similarity). Richness estimates were highest in the sediment, biofilms, and benthic samples (189-231 OTUs), intermediate in water samples (42-85 OTUs), and lowest in plankton samples (8 OTUs). NMDS grouped the eight studied habitats into six clusters, indicating that community composition was strongly influenced by turnover among habitats. Fungal communities exhibited changes at the phylum and order levels along three different substrate categories from littoral to pelagic habitats. The large majority of OTUs (> 75%) could not be classified below the order level due to the lack of aquatic fungal entries in public sequence databases. Our study provides a first estimate of lake-wide fungal diversity and highlights the important contribution of habitat heterogeneity to overall diversity and community composition. Habitat diversity should be considered in any sampling strategy aiming to assess the fungal diversity of a water body.
Environmental actinorhodopsin expression revealed by a new in situ filtration and fixation sampler
(2012)
Freshwater Actinobacteria are an important and dominant group of bacterioplankton in most temperate freshwater systems. Recently, metagenomic studies discovered rhodopsin-like protein-coding sequences present in Actinobacteria which could be a decisive hint for their success in freshwater ecosystems. We analysed the diversity of actinorhodopsin (ActR) in Lake Stechlin (northern Germany) and assessed the actR expression profile during a diurnal cycle. We obtained 85 positive actR clones which could be subsequently grouped to 17 operational taxonomic units assuming a 90% sequence similarity. The phylogenetic analysis points to a close relationship of all obtained sequences to the acI lineage of Actinobacteria, forming six independent clusters. For the first time, we followed in situ transcription of actR in Lake Stechlin revealing a rather constitutive circadian gene expression. For analysing in situ expression patterns of functional genes in aquatic ecosystems, such as actR, we invented a new in situ filtration and fixation sampler (IFFS). The IFFS enables the representative investigation of microbial transcriptomes in any aquatic ecosystem at all water depths. The IFFS sampler is simple and inexpensive, and we provide all engineering plans for an easy rebuild. Consequently, our IFFS is suitable to reliably study expression of any known functional gene of any aquatic microorganism.
Fungi and bacteria are the major organic matter (OM) decomposers in aquatic ecosystems. While bacteria are regarded as primary mineralizers in the pelagic zone of lakes and oceans, fungi dominate OM decomposition in streams and wetlands. Recent findings indicate that fungal communities are also active in lakes, but little is known about their diversity and interactions with bacteria. Therefore, the decomposer niche overlap of saprotrophic fungi and bacteria was studied on pollen (as a seasonally recurring source of fine particulate OM) by performing microcosm experiments with three different lake types. Special emphasis was placed on analysis of fungal community composition and diversity. We hypothesized that (I) pollen select for small saprotrophic fungi and at the same time for typical particle-associated bacteria; (II) fungal communities form specific free-living and attached sub-communities in each lake type; (III) the ratio between fungi or bacteria on pollen is controlled by the lake's chemistry. Bacteria-to-fungi ratios were determined by quantitative PCR (qPCR), and bacterial and fungal diversity were studied by clone libraries and denaturing gradient gel electrophoresis (DGGE) fingerprints. A protease assay was used to identify functional differences between treatments. For generalization, systematic differences in bacteria-to-fungi ratios were analyzed with a dataset from the nearby Baltic Sea rivers. High abundances of Chytridiomycota as well as occurrences of Cryptomycota and yeast-like fungi confirm the decomposer niche overlap of saprotrophic fungi and bacteria on pollen. As hypothesized, microbial communities consistently differed between the lake types and exhibited functional differences. Bacteria-to-fungi ratios correlated well with parameters such as organic carbon and pH. The importance of dissolved organic carbon and nitrogen for bacteria-to-fungi ratios was supported by the Baltic Sea river dataset. Our findings highlight the fact that carbon-to-nitrogen ratios may also control fungal contributions to OM decomposition in aquatic ecosystems.
Shifts among Eukaryota, Bacteria, and Archaea define the vertical organization of a lake sediment
(2017)
Background: Lake sediments harbor diverse microbial communities that cycle carbon and nutrients while being constantly colonized and potentially buried by organic matter sinking from the water column. The interaction of activity and burial remained largely unexplored in aquatic sediments. We aimed to relate taxonomic composition to sediment biogeochemical parameters, test whether community turnover with depth resulted from taxonomic replacement or from richness effects, and to provide a basic model for the vertical community structure in sediments. Methods: We analyzed four replicate sediment cores taken from 30-m depth in oligo-mesotrophic Lake Stechlin in northern Germany. Each 30-cm core spanned ca. 170 years of sediment accumulation according to Cs-137 dating and was sectioned into layers 1-4 cm thick. We examined a full suite of biogeochemical parameters and used DNA metabarcoding to examine community composition of microbial Archaea, Bacteria, and Eukaryota. Results: Community beta-diversity indicated nearly complete turnover within the uppermost 30 cm. We observed a pronounced shift from Eukaryota- and Bacteria-dominated upper layers (<5 cm) to Bacteria-dominated intermediate layers (5-14 cm) and to deep layers (>14 cm) dominated by enigmatic Archaea that typically occur in deep-sea sediments. Taxonomic replacement was the prevalent mechanism in structuring the community composition and was linked to parameters indicative of microbial activity (e.g., CO2 and CH4 concentration, bacterial protein production). Richness loss played a lesser role but was linked to conservative parameters (e.g., C, N, P) indicative of past conditions. Conclusions: By including all three domains, we were able to directly link the exponential decay of eukaryotes with the active sediment microbial community. The dominance of Archaea in deeper layers confirms earlier findings from marine systems and establishes freshwater sediments as a potential low-energy environment, similar to deep sea sediments. We propose a general model of sediment structure and function based on microbial characteristics and burial processes. An upper "replacement horizon" is dominated by rapid taxonomic turnover with depth, high microbial activity, and biotic interactions. A lower "depauperate horizon" is characterized by low taxonomic richness, more stable "low-energy" conditions, and a dominance of enigmatic Archaea.
Most studies of aquatic plankton focus on either macroscopic or microbial communities, and on either eukaryotes or prokaryotes. This separation is primarily for methodological reasons, but can overlook potential interactions among groups. Here we tested whether DNA metabarcoding of unfractionated water samples with universal primers could be used to qualitatively and quantitatively study the temporal dynamics of the total plankton community in a shallow temperate lake. Significant changes in the relative proportions of normalized sequence reads of eukaryotic and prokaryotic plankton communities over a 3-month period in spring were found. Patterns followed the same trend as plankton estimates measured using traditional microscopic methods. The bloom of a conditionally rare bacterial taxon belonging to Arcicella was characterized, which rapidly came to dominate the whole lake ecosystem and would have remained unnoticed without metabarcoding. The data demonstrate the potential of universal DNA metabarcoding applied to unfractionated samples for providing a more holistic view of plankton communities.
Citizen science projects have a long history in ecological studies. The research usefulness of such projects is dependent on applying simple and standardized methods. Here, we conducted a citizen science project that involved more than 3500 Swedish high school students to examine the temperature difference between surface water and the overlying air (T-w-T-a) as a proxy for sensible heat flux (Q(H)). If Q(H) is directed upward, corresponding to positive T-w-T-a, it can enhance CO2 and CH4 emissions from inland waters, thereby contributing to increased greenhouse gas concentrations in the atmosphere. The students found mostly negative T-w-T-a across small ponds, lakes, streams/rivers and the sea shore (i.e. downward Q(H)), with T-w-T-a becoming increasingly negative with increasing T-a. Further examination of T-w-T-a using high-frequency temperature data from inland waters across the globe confirmed that T-w-T-a is linearly related to T-a. Using the longest available high-frequency temperature time series from Lake Erken, Sweden, we found a rapid increase in the occasions of negative T-w-T-a with increasing annual mean T-a since 1989. From these results, we can expect that ongoing and projected global warming will result in increasingly negative T-w-T-a, thereby reducing CO2 and CH4 transfer velocities from inland waters into the atmosphere.
In this pilot study, we describe a high-pressure incubation system allowing multiple subsampling of a pressurized culture without decompression. The system was tested using one piezophilic (Photobacterium profundum), one piezotolerant (Colwellia maris) bacterial strain and a decompressed sample from the Mediterranean deep sea (3044 m) determining bacterial community composition, protein production (BPP) and cell multiplication rates (BCM) up to 27 MPa. The results showed elevation of BPP at high pressure was by a factor of 1.5 +/- 1.4 and 3.9 +/- 2.3 for P. profundum and C. maris, respectively, compared to ambient-pressure treatments and by a factor of 6.9 +/- 3.8 fold in the field samples. In P. profundum and C. maris, BCM at high pressure was elevated (3.1 +/- 1.5 and 2.9 +/- 1.7 fold, respectively) compared to the ambient-pressure treatments. After 3 days of incubation at 27 MPa, the natural bacterial deep-sea community was dominated by one phylum of the genus Exiguobacterium, indicating the rapid selection of piezotolerant bacteria. In future studies, our novel incubation system could be part of an isopiestic pressure chain, allowing more accurate measurement of bacterial activity rates which is important both for modeling and for predicting the efficiency of the oceanic carbon pump.
Pelagic bacteria can be classified into free-living and particle-attached life modes, which either dwell in the water column or attach to suspended particles. Bacteria with a generalist life style, however, can actively shift between these two habitats. Globally increasing densities of natural and artificial particles enhance habitat heterogeneity, with potential consequences for system stability and trophic transfer through aquatic food webs. To better decipher the dynamics of microbial communities, we investigated the influence of adaptive vs. fixed habitat choice on species coexistence for a simplified bacterial community by analyzing a corresponding food web model, consisting of two specialist bacterial prey species (free and attached), a generalist bacterial prey species with the ability to shift between both habitats, and two protist predators, specialized on either water or particle compartment. For simplicity we assume a shared resource pool, considering particles only for colonization but not as a source for nutrients or carbon, that is, inert particles like microplastics or inorganic sediments. The model predicts coexistence on a cyclic attractor between fixed and flexible bacteria, if the costs for adaptive habitat choice can be balanced by adaptation speed. The presence of adaptive prey dampens predator-prey cycle amplitudes, contributing to system stabilization resulting in higher mean predator biomass compared to specialist prey only. Thus, in pelagic microbial systems, flexible habitat choice at the prey level has important implications for system stability and magnitude of energy flow through the microbial loop.