Exploring the use of fish as indicators of eicosapentaenoic and docosahexaenoic supply in lake ecosystems.

An adequate supply of food sources with high levels (i.e., weight proportion of total fatty acids) and contents (i.e., absolute amount per mass) of long chain polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are important for ecosystems. Therefore, the supply of EPA and DHA from basal food sources is a useful indicator of ecosystem health. To determine whether EPA and DHA levels and contents in fish can be used as indicators of EPA and DHA supply in lakes, five dominant species of fish and basal food sources (seston and sediment) were collected monthly from June to November from 2016 to 2021 from Lake Hachiro, Japan. Seston and Hypomesus nipponensis were collected from 12 lakes (one collection per lake) with varying seston contents in EPA and DHA. The trends of EPA and DHA in all fish species were similar to those of the basal food sources. Correlation analysis showed that the EPA levels were strongly correlated between fish and seston; moreover, the correlation coefficient increased when a 1- or 2-month moving average was applied to the basal food sources, suggesting that fish represent a time-integrated supply of EPA and DHA. EPA levels of H. nipponensis had the highest correlation coefficients with seston among all fish species. EPA levels of H. nipponensis were significantly correlated with those of seston among lakes. The results of this study suggest that H. nipponensis is a useful indicator of EPA and DHA supplies in lake ecosystems.

Sulfuricystis multivorans gen. nov., sp. nov. and Sulfuricystis thermophila sp. nov., facultatively autotropic sulfur-oxidizing bacteria isolated from a hot spring, and emended description of the genus Rugosibacter.

Strains J5BT and M52T are facultatively autotrophic sulfur-oxidizing bacteria isolated from a microbial mat from a hot spring. They were isolated and partially characterized in previous studies, as facultative anaerobes which use nitrate as electron acceptor. In this study, additional characterizations were made to determine their taxonomic status. In both strains, major cellular fatty acids were C16:1 (C16:1ω7c and/or C16:1ω6c) and C16:0. Their chemolithoautotrophic growth was supported by thiosulfate and elemental sulfur. They used some organic acids as growth substrates. Their 16S rRNA gene sequences indicated the highest sequence identities to species in the family Sterolibacteriaceae, but the identities were 95% or lower. Phylogenetic analysis indicated that these strains do not belong to any existing genera. Values of average nucleotide identity and digital DNA-DNA hybridization between strains J5BT and M52T were 87.93% and 34.3%, respectively. On the basis of phenotypic and genomic characteristics, Sulfuricystis multivorans gen. nov. sp. nov., and Sulfuricystis thermophila sp. nov. are proposed, with type strains of J5BT and M52T, respectively. An emended description of the genus Rugosibacter is also proposed, for its reclassification to the family Sterolibacteriaceae.

Desulfofustis limnaeus sp. nov., a freshwater sulfate-reducing bacterium isolated from marsh soil.

A novel sulfate-reducing bacterium, strain PPLLT, was isolated from marsh soil. Cells of strain PPLLT were rod-shaped with length of 1.5 µm and width of 0.7 µm. Growth was observed at 22-37 °C (optimum 35 °C) and pH 6.8-8.4 (optimum 7.3). Lactate, succinate, fumarate, formate and malate were utilized as electron donors for sulfate reduction. Fermentative growth was not observed on tested organic acids. Besides sulfate, sulfite, thiosulfate and elemental sulfur were utilized as electron acceptors. Hydrogen is used only in the presence acetate or yeast extract. The major fatty acid was C16:0. The complete genome of strain PPLLT was composed of a circular chromosome with length of 4.2 Mbp and G + C content of 57.7 mol%. Sequence analysis of the 16S rRNA gene showed that strain PPLLT was affiliated with the genus Desulfofustis in the family Desulfocapsaceae. On the basis of differences in the phylogenetic and phenotypic properties between the strain and the type strain of the genus Desulfofustis, strain PPLLT (DSM 110475T = JCM 39161T) is proposed as the type strain of a new species, with name of Desulfofustis limnaeus sp. nov.

Transfer of cyanobacterial carbon to a higher trophic-level fish community in a eutrophic lake food web: fatty acid and stable isotope analyses.

The dietary utilization of cyanobacterial carbon by fish communities is poorly understood. We examined the transfer of cyanobacterial carbon to fish in a eutrophic lake using fatty acid biomarkers and measuring the stable carbon isotope ratios of fatty acid and bulk nitrogen. We collected five species of fish (Hypomesus nipponensis, Carassius sp., Cyprinus carpio, Tridentiger brevispinis, and Gymnogobius castaneus) as well as the seston from June to November 2016 from Lake Hachiro, Japan. Cyanobacterial blooms were observed from August to October. From June to August, cyanobacterial fatty acid biomarkers (18:2ω6 and 18:3ω3) accounted for only 1.4-4.3% of total fatty acids in these fish species, indicating a low contribution of cyanobacteria to fish diets during this period. However, the contribution of the cyanobacterial fatty acid biomarkers in these fish species increased sharply in September (10.5-17.1%), except in second-year H. nipponensis. In September, the stable carbon isotope ratios of 18:3ω3 in these fish species were almost equivalent to those in the seston, which was primarily composed of cyanobacteria. The trophic positions of the collected fish species ranged from 1.6 to 3.4, based on their stable nitrogen isotope values, indicating that some fish ingested cyanobacteria directly, while others acquired cyanobacteria indirectly, through the food chain. These findings indicate that cyanobacterial carbon is transferred up the food chain in eutrophic lake ecosystems with cyanobacterial blooms.

Fungal mn oxides supporting Mn(II) oxidase activity as effective Mn(II) sequestering materials.

We examined the Mn(II)-oxidizing ability of the biogenic Mn oxide (BMO) formed in cultures ofa Mn(II)-oxidizing fungus, Acremonium strictum strain KR21-2. The newly formed BMO effectively sequestered dissolved Mn(II) mainly by oxidizing Mn(II) to insoluble Mn under air-equilibrated conditions, and this ability lasted for at least 8 days. Deaerating the BMOs, poisoning them with NaN3, or heating them all readily weakened their Mn(II) oxidation ability, indicating the involvement of enzymatic Mn(II) oxidation. There was no Mn(II)-oxidizing ability observed for mycelia cultivated without Mn(II) or for residual mycelia after the BMO phase was dissolved, suggesting the need for the oxide phase. A sodium dodecyl sulphate-polyacrylamide gel electrophoresis assay demonstrated that the oxide phase embeds the Mn(II) oxidase and thereby maintains the enzymatic activity in BMOs. Freezing at -80 degrees C preserved the Mn(II)-oxidizing ability in BMOs for at least 4 weeks, while lyophilization caused a complete loss of this ability. Based on these results, we propose that fungal Mn oxides supporting Mn(II) oxidase activity are an effective Mn(II)-sequestering material capable of oxidizing Mn(II) continuously from solutions containing no additional nutrients to maintain biological activity.

Effects of Algal Extracellular Polysaccharides on the Formation of Filamentous Manganese Oxide Particles in the Near-Bottom Layer of Lake Biwa.

Filamentous manganese (Mn) oxide particles, which occur in the suboxic zone of stratified waterbodies, are important drivers of diverse elemental cycles. These particles are considered to be bacteriogenic; despite the importance of biogeochemical implications, however, the environmental factor responsible for their formation has not been identified. The aim of this study was to demonstrate the involvement of algal extracellular polysaccharides in Mn oxide particle formation. Based on this study of laboratory cultures of a model Mn(II)-oxidizing bacterium, the supply of algal extracellular mucilage was shown to stimulate Mn(II) oxidation and thus the production of filamentous Mn oxide particles. This observation was consistent with the results obtained for naturally occurring particles collected from a near-bottom layer (depth of approximately 90 m) in the northern basin of Lake Biwa, Japan, that is, most Mn particles resembling δ-MnO2 were associated with an extracellular mucilage-like gelatinous matrix, which contained dead algal cells and was lectin-stainable. In the lake water column, polysaccharides produced by algal photosynthesis sank to the bottom layer. The analysis of the quality of water samples, which have been collected from the study site for 18 years, reveals that the annual average total phytoplankton biovolume in the surface layer correlates with the density of filamentous Mn particles in the near-bottom layer. Among different phytoplankton species, green algae appeared to be the key species. The results of this study suggest that algal extracellular polysaccharides serve as an important inducer for the formation of filamentous Mn oxide particles in the near-bottom layer of the northern basin of Lake Biwa.

Isolation of a Leptothrix strain, OUMS1, from ocherous deposits in groundwater.

Leptothrix species in aquatic environments produce uniquely shaped hollow microtubules composed of aquatic inorganic and bacterium-derived organic hybrids. Our group termed this biologically derived iron oxide as "biogenous iron oxide (BIOX)". The artificial synthesis of most industrial iron oxides requires massive energy and is costly while BIOX from natural environments is energy and cost effective. The BIOX microtubules could potentially be used as novel industrial functional resources for catalysts, adsorbents and pigments, among others if effective and efficient applications are developed. For these purposes, a reproducible system to regulate bacteria and their BIOX productivity must be established to supply a sufficient amount of BIOX upon industrial demand. However, the bacterial species and the mechanism of BIOX microtubule formation are currently poorly understood. In this study, a novel Leptothrix sp. strain designated OUMS1 was successfully isolated from ocherous deposits in groundwater by testing various culture media and conditions. Morphological and physiological characters and elemental composition were compared with those of the known strain L. cholodnii SP-6 and the differences between these two strains were shown. The successful isolation of OUMS1 led us to establish a basic system to accumulate biological knowledge of Leptothrix and to promote the understanding of the mechanism of microtubule formation. Additional geochemical studies of the OUMS1-related microstructures are expected provide an attractive approach to study the broad industrial application of bacteria-derived iron oxides.

Origin of Carbon and Essential Fatty Acids in Higher Trophic Level Fish in Headwater Stream Food Webs.

Dietary carbon sources in headwater stream food webs are divided into allochthonous and autochthonous organic matters. We hypothesized that: 1) the dietary allochthonous contribution for fish in headwater stream food webs positively relate with canopy cover; and 2) essential fatty acids originate from autochthonous organic matter regardless of canopy covers, because essential fatty acids, such as 20:5ω3 and 22:6ω3, are normally absent in allochthonous organic matters. We investigated predatory fish Salvelinus leucomaenis stomach contents in four headwater stream systems, which are located in subarctic region in northern Japan. In addition, stable carbon and nitrogen isotope ratios, fatty acid profile, and stable carbon isotope ratios of essential fatty acids were analyzed. Bulk stable carbon analysis showed the major contribution of autochthonous sources to assimilated carbon in S. leucomaenis. Surface baits in the stomach had intermediate stable carbon isotope ratios between autochthonous and allochthonous organic matter, indicating aquatic carbon was partly assimilated by surface baits. Stable carbon isotope ratios of essential fatty acids showed a positive relationship between autochthonous sources and S. leucomaenis across four study sites. This study demonstrated that the main supplier of dietary carbon and essential fatty acids was autochthonous organic matter even in headwater stream ecosystems under high canopy cover.

Microbial manganese oxide formation and interaction with toxic metal ions.

Diverse bacteria and fungi oxidize Mn(II) enzymatically and produce insoluble Mn(III, IV) oxides, and these organisms are considered to be the primal agents for the occurrence of natural Mn oxide phases in most environments. Biogenic Mn oxides have a high sorption capacity for metal cations and an ability to oxidize numerous inorganic and organic compounds, owing to their structural and redox features. Thus, the microbial process is of significance in both biogeochemical and biotechnological contexts. In this article we summarize the enzymatic Mn(II) oxidation and interactions of biogenic Mn oxides with toxic metal and metalloid ions. Although Mn oxide formation by fungi has not been fully characterized yet, recent researches with ascomycetes emphasize the similarity between the bacterial and fungal Mn(II) oxidation with respect to the involved catalyst (i.e., multicopper oxidase-type enzymes) and the reaction product [i.e., layer-type Mn(IV) oxides]. Laboratory cultures of bacterial and fungal Mn oxidizers are expected to provide fundamental knowledge in their potential use for remediation of environments and effluents contaminated with toxic metal(loid) ions.

Production of biogenic manganese oxides by repeated-batch cultures of laboratory microcosms.

We investigated the production of manganese (Mn) oxides using repeated-batch bioreactors maintained over long periods under laboratory conditions. Freshwater epilithic biofilms were used as the initial inocula. The bioreactors yielded suspended solids that could remove 0.1 mM dissolved Mn(II) within a few days. Chemical titration, X-ray absorption near-edge structure spectroscopy, and X-ray diffraction analysis revealed that the Mn(II) had been converted to poorly crystallized layer-type Mn(IV) oxides, which were similar to known biogenic Mn oxides from pure bacterial cultures. Spherical or rod-shaped Mn microconcretions occurred in the suspended solids; transmission electron microscopy showed that these structures likely resulted from the microbial activity but not represent living cells. Instead, the presence of encapsulated, sheathed, and hyphal budding cells in the suspended solids indicated that a range of Mn-depositing bacteria contributed to the Mn oxide formation. To our knowledge, our data represent the first observation of production of such Mn oxides in a laboratory microcosm wherein a range of Mn-depositing bacteria coexist. The fact that sorption of trace Zn(II) and Ni(II) ions onto the suspended solids co-occurred with the removal of dissolved Mn(II) emphasizes the important role of Mn-oxidizing microorganisms in the fates of trace or contaminant metals in the aquatic environment.

Whole-Genome Sequences of Two Manganese(II)-Oxidizing Bacteria, Bosea sp. Strain BIWAKO-01 and Alphaproteobacterium Strain U9-1i.

This report describes the whole-genome sequences of two Mn(II)-oxidizing bacteria, filamentous Mn oxide microparticle-forming Bosea sp. strain BIWAKO-01 and alphaproteobacterium strain U9-1i.

Whole Genome Sequence of the Non-Microcystin-Producing Microcystis aeruginosa Strain NIES-44.

Microcystis aeruginosa is a typical algal bloom-forming cyanobacterium. This report describes the whole-genome sequence of a non-microcystin-producing strain of Microcystis aeruginosa, NIES-44, which was isolated from a Japanese lake.

Complete Genome Sequence of the Unclassified Iron-Oxidizing, Chemolithoautotrophic Burkholderiales Bacterium GJ-E10, Isolated from an Acidic River.

Burkholderiales bacterium GJ-E10, isolated from the Tamagawa River in Akita Prefecture, Japan, is an unclassified, iron-oxidizing chemolithoautotrophic bacterium. Its single circular genome, consisting of 3,276,549 bp, was sequenced by using three types of next-generation sequencers and the sequences were then confirmed by PCR-based Sanger sequencing.

Application of Euglena gracilis cells to comet assay: evaluation of DNA damage and repair.

Alkaline single-cell gel electrophoresis (comet assay) enables sensitive detection of DNA damage in eukaryotic cells induced by genotoxic agents. We performed a comet assay of unicellular green alga Euglena gracilis that was exposed to genotoxic chemicals, 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), benzo[a]pyrene (BAP), mitomycin C (MMC) and actinomycin D (AMD). Tail length and tail moment in migrated DNA were measured as indications of DNA damage. MNNG and BAP were found to cause concentration-dependent increases in DNA damage. The responses were more sensitive than those of human lymphocytes under the same treatment conditions. MMC and AMD showed no positive response, as reported elsewhere. The comet assays performed at specified times after treatment revealed that the DNA damaged by MNNG and gamma-ray irradiation was repaired during the initial 1h. The results clearly show that the comet assay is useful for evaluating chemically-induced DNA damage and repair in E. gracilis. Given the ease of culturing and handling E. gracilis as well as its sensitivity, the comet assay of this alga would undoubtedly prove to be a useful tool for testing the genotoxicity of chemicals and monitoring of environmental pollution.

Growth of Aspergillus oryzae during treatment of cassava starch processing wastewater with high content of suspended solids.

Aspergillus oryzae IFO 30113 was used for the treatment of the cassava starch processing (CSP) wastewater. The observations on the fungal morphology showed that, in the shake flasks containing the CSP wastewater with the high concentration of suspended solids, the formation of pellets originated from the adherence of germinated spores to solid particles in medium. The attached solid particles were also digested during the fungal fermentation and resulted in the formation of the smooth and hollow pellets. The changes of the culture conditions such as inoculum size, initial pH of wastewater, inoculum type and nutrient elements affected on the fungal morphology, biomass accumulation and treatment efficiencies of A. oryzae IFO 30113. In the typical pH range (pH 4-5) of the CSP wastewater, the formation of smooth pellets was predominant and A. oryzae IFO 30113 was satisfiable for the production of fungal biomass and treatment efficiencies. The supplementation of nitrogen sources has shown an improvement in the fungal biomass accumulation and the treatment efficiency of A. oryzae IFO 30113 growing in the CSP wastewater. Especially, high biomass yields (up to 0.8 g/g-COD) were achieved in flasks supplied with peptone. With ammonium sulfate as nitrogen source, 87% total organic carbon (TOC), 91% COD and 94% starch were removed after 96-h incubation. The possibility of the pellet formation despite the presence of the high content of suspended solids would be of great advantage to perform the treatment process and the fungal biomass production on the airlift-type bioreactors by lowering medium viscosity and better mass exchange of oxygen and nutrients.

Anaerobic degradation of cis-1,2-dichloroethylene and vinyl chloride by Clostridium sp. strain DC1 isolated from landfill leachate sediment.

The bacterial community structure of anaerobic enrichment cultures that are capable of degrading both cis-1,2-dichloroethylene (cis-DCE) and vinyl chloride (VC) and isolation of the organism responsible for the degradation were investigated. Denaturing gradient gel electrophoresis (DGGE) of a PCR-amplified 16S rRNA gene from the cultures showed the possible predominance of Clostridium species. One isolate, designated strain DC1, was closely related to members of Clostridiaceae, based on 16S rRNA gene analysis, and the highest sequence similarity (98.9%) was obtained for Clostridium saccarobutylicum. In culture experiments, strain DC1 was shown to degrade cis-DCE and VC during the stationary phase of growth without accumulation of VC and/or ethene. The bacterial growth was not linked to the degradation of cis-DCE and VC. Stoichiometric analysis revealed that two moles of chloride ions as released from one mole of cis-DCE during the incubation period, indicating that cis-DCE was fully dechlorinated. The results appear consistent with the presence of a mechanism of oxidative dechlorination rather than respiratory reductive dechlorination; the latter is accompanied by transient formation of dechlorinated ethenes from cis-DCE and VC.

Removal of heavy metal cations by biogenic magnetite nanoparticles produced in Fe(III)-reducing microbial enrichment cultures.

The biogenic magnetite nanoparticles presented here had a high capacity of adsorbing metal cations, which was approximately 30- to 40-fold greater than commercially available magnetite. These results suggest the potential application of microbial magnetite formation in the removal of toxic metal cations from water.

Manganese(IV) oxide production by Acremonium sp. strain KR21-2 and extracellular Mn(II) oxidase activity.

Ascomycetes that can deposit Mn(III, IV) oxides are widespread in aquatic and soil environments, yet the mechanism(s) involved in Mn oxide deposition remains unclear. A Mn(II)-oxidizing ascomycete, Acremonium sp. strain KR21-2, produced a Mn oxide phase with filamentous nanostructures. X-ray absorption near-edge structure (XANES) spectroscopy showed that the Mn phase was primarily Mn(IV). We purified to homogeneity a laccase-like enzyme with Mn(II) oxidase activity from cultures of strain KR21-2. The purified enzyme oxidized Mn(II) to yield suspended Mn particles; XANES spectra indicated that Mn(II) had been converted to Mn(IV). The pH optimum for Mn(II) oxidation was 7.0, and the apparent half-saturation constant was 0.20 mM. The enzyme oxidized ABTS [2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] (pH optimum, 5.5; Km, 1.2 mM) and contained two copper atoms per molecule. Moreover, the N-terminal amino acid sequence (residues 3 to 25) was 61% identical with the corresponding sequence of an Acremonium polyphenol oxidase and 57% identical with that of a Myrothecium bilirubin oxidase. These results provide the first evidence that a fungal multicopper oxidase can convert Mn(II) to Mn(IV) oxide. The present study reinforces the notion of the contribution of multicopper oxidase to microbially mediated precipitation of Mn oxides and suggests that Acremonium sp. strain KR21-2 is a good model for understanding the oxidation of Mn in diverse ascomycetes.

Enzymatic formation of manganese oxides by an Acremonium-like hyphomycete fungus, strain KR21-2.

A Mn-depositing fungus, Acremonium-like hyphomycete strain KR21-2, was isolated from a Mn deposit occurring on the wall of a storage bottle containing Mn(III, IV) oxide-coated streambed pebbles and stream water. 18S rRNA gene sequence analysis revealed that strain KR21-2 was phylogenetically related to members of the order Hypocreales within the class Ascomycetes. The spent culture medium at the stationary phase of fungal growth contained a 54-kDa protein capable of depositing Mn oxides. The enzymatic activity was inhibited by azide and o-phenanthroline. The Mn(II)-oxidizing protein possessed a laccase activity, as indicated by direct oxidation of p-phenylenediamine and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid). These results are consistent with the role assumed for laccase-like multicopper oxidase, which is proposed to be involved in the Mn(II)-oxidizing factors from some bacteria. Unlike laccases of basidiomycete fungi, however, the protein of strain KR21-2 did not produce soluble Mn(III) species in the presence of either of the Mn chelators pyrophosphate and malonate. This is the first report on the possible involvement of laccase and/or multicopper oxidase in Mn oxide deposition by ascomycetes (including their anamorphs) ubiquitous in natural environments.