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Microalgae-bacteria symbiotic association can provide many competitive advantages over the pure axenic culture of microalgae to obtain biofuel. This system significantly reduces an important technical constraint, including associated capital cost with the infrastructure required for maintaining pure microalgae culture. The current study aims to compare the symbiotic growth of Tetraselmis indica - Pseudomonas aeruginosa consortium with axenic growth of Tetraselmis indica in dairy wastewater (DWW). The extraction and optimization of chlorophyll-a were also investigated. The overall maximum chlorophyll-a recovery of 29.106 ± 0.045 mg L−1 was obtained through the sonification as extractant method with methanol as an extraction solvent. The chlorophyll-a content of consortium cultivated in DWW higher than the axenic pure microalgae culture (APMC). The maximum biomass yield of the consortium was achieved as 1454.88 mg L−1, which is much higher than APMC growth in DWW by 38.80%. On the 10th day, the consortium removed 87.49%, 83.76%, and 79.83% of chemical oxygen demand (COD), total dissolved nitrogen (TDN), and total dissolved phosphorus respectively. The higher biomass growth and wastewater nutrient removal exhibited by the symbiotic microalgae-bacteria consortium can be utilized in biofuel technology.

Microalgae produce a broad range of organic compounds that are increasingly being recognised for their value in novel product production and biotechnological applications. Most microalgae are photoautotrophic, but some are capable of either mixotrophy or heterotrophy. Reported enhanced biomass yields or contrasting metabolite profiles compared to autotrophic growth improve the economics of large-scale production of microalgae, which currently limits industrial applications. Here, the potential of a high-throughput method for the rapid screening of microalgal metabolism was assessed against 95 different carbon sources, using the cost-effective Biolog plate. Of the 5 microalgae tested, Desmodesmus communis (30 carbon sources) and Chlorella vulgaris (19 carbon sources) had the highest number of positive responses to carbon sources, whereas Chlorella sorokiniana had the most negative (toxic) response to the various carbon sources (77 carbon sources). Comparison of Biolog plate results with traditional culture techniques showed good agreement. Species with a high number of positive responses on the Biolog plate exhibited the highest biomass yield under heterotrophic conditions, whilst those with low number of positive responses exhibited the highest biomass yield under autotrophic conditions, using traditional culturing techniques. While the use of these plates is limited to obtaining axenic lines of microalgal species, the method provided a high-throughput assessment of carbon source metabolism, without the expense of undertaking large, laborious traditional culturing assessments. Such high-throughput assessments can be regarded as useful tools for progressing species selection, metabolic capacity and optimal culture conditions for microalgal biotechnology applications.

Endophytic fungi Cladosporium cladosporioides (F1-MH810309) and Cladosporium tenuissimum (F2-MN715834) from the leaf of wild Cymbopogon martinii (MT90507) were isolated and selected based on the persistent occurrence during different seasons of the year. They were identified based on the morphological features and molecular characterization (ITS sequence), and later deposited at NCBI. Phytochemical studies on F1, F2 and host extracts showed the presence of alkaloids, flavonoids, phenols, terpenoids and tannins. The GC-MS of F1 extract (control) under the axenic condition revealed compounds like hexadecane, heptadecane,2,4-Ditert-butylphenol, E-14 hexadecenal, geraniol, geranyl acetate and cubenol similar to the host. The GC-MS of F2 extract (control) revealed metabolites that were unique. Further, both F1 and F2 were cultured in the supplementation of different concentrations (5%, 10%, 15% and 20%) of the host plant extract (an-axenic condition). The GC-MS of F1 extracts (test) exhibited good growth and showed the gradual increased production of terpenoid compounds whereas the F2 (test) did not show any growth. These compounds such as hyrdoxymenthol, nor-borneol, cedralacetate, α-cyclogeraniol, campesterol, β-cyclogeraniol, linalool oxide,2,3-boranediol, citronellyltiglate and 2,3-pinanediol were produced in a minor quantity and were known as biotransformed forms of the precursor compounds present in the host extract. In comparison, only F1 was able to produce terpenoids similar to the host species both in axenic and an-axenic conditions. Hence from the current study, the endophytic fungus F1 isolated from wild C. martinii for the first time can serve as a better resource for the bioprospection of an important terpenoid and its metabolites.

Microalgae are emerging sources for sustainable production of bioproducts and bioenergy. However, a problem with its reliable production is the biological contamination of cultures. There are several alternatives to mitigate this problem, such as using axenic cultures, concentrated inocula, or applying chemical control agents, which should be eco-friendly. Neochloris oleoabundans is an oleaginous green microalga with a high potential for biofuel production due to its ability to accumulate neutral lipids, mainly monounsaturated fatty acids. For this reason, the main objective of this article was to evaluate the toxicity of 6 natural terpenes with proven antibacterial activity (α-pinene, β-pinene, limonene, trans-cinnamaldehyde, linalool, and eugenol) towards cultures of this green microalgae, through toxicity analysis to obtain the lethal concentration 50 (LC50). The values for β-pinene and limonene were 17 and 16 ppm, respectively, lower than the levels obtained for α-pinene and trans-cinnamaldehyde, of 65 and 35 ppm, respectively. Eugenol and linalool showed LC50 levels above 250 ppm. These results show that selected terpenes do not present high toxicity against N. oleoabundans. Therefore, they can be a green alternative in the management of the cultures.

Endophytic fungi have demonstrated a harmless relationship living within the internal tissues of several plant hosts and at the same time produce diverse important bioactive compounds having a wide range of pharmaceutical applicability. In this study, nine endophytic fungi, eight from Cassava leaf:Clg1, Clg2, Clr4, Clr5, Clr6, Clr7,Clr9and one from Cassava mid-rib:CRs3with distinct cultural features were isolated from healthy leaves ofManihot utilisimaand axenic cultures were fermented on sterile rice medium for 21 days. The antimicrobial and antioxidant activities of the fungal crude extracts were evaluated. Chemical analyses of the metabolic profiles of each fungal extract revealed the presence of nine known compounds with established biological activities. Each fungal extract exhibited antimicrobial activities against at least one Gram positive and Gram negative bacteria with an inhibition zone that ranged from 2 – 6 mm.Clg2andCRs3fungal extracts demonstrated moderate potential to scavenge free radical with an inhibition of 58 and 60% respectively. Septicine, cyclo(prolylvalyl), pentenedioc acid, neurolenin B, rubrofusarin, p-Hydroxybenzoic acid, protocatechuic acid, citreoisocoumarin, palitantin and pestalio pyrone were the compounds detected in the fungal fermentation products. Our findings reveal thatM. utilisimaleaves harbor endophytic fungi with unique chemodiversity of bioactive secondary metabolites needed for development of new drugs.&nbsp

This study aims to evaluate the anti-Leishmania major and the lung adenocarcinoma (A549) cytotoxicity of Withania somnifera root and fruit. The total extracts were obtained by homogenisation in aqueous MeOH, and the sub-extracts [n-hexane, ethyl acetate (EtOAc), n-butanol (n-BuOH), and methanol (MeOH)] were obtained by flash chromatography. The activity evaluation showed that n-BuOH sub-extracts from root and fruit exhibited noticeable antileishmanial promastigote properties. The n-hexane and EtOAc sub-extracts from both organs, and the MeOH sub-extract from the fruit exerted mild to moderate effects on the promastigotes. In-vitro growth-inhibitory test results on axenic amastigote and cytotoxicity testing on macrophages (RAW264.7), the parasite-host at the amastigote stage, revealed that the activity was mainly concentrated in the root EtOAc and n-BuOH sub-extracts and to a lesser extent the fruit MeOH and EtOAc, and the root n-hexane sub-extracts. Only the roots' EtOAc and n-BuOH sub-extracts demonstrated low cytotoxicity on the A549 cell line.

Leishmaniasis is one of the most neglected tropical diseases in the world, causing high mortality and morbidity. In addition, the medications used to treat this disease are highly toxic, long-term used, and with resistance problems. This emphasizes the need to search for new molecules with antileishmanial potential. In the present work, the methanol extract from the rhizomes of Dorstenia contrajerva L., Moraceae and the compounds cycloartocarpesin (1) and 1-O-linolenoyl-2-O-stearoyl-3-O-β-d-galactopyranosyl glycerol (2) were evaluated against axenic amastigotes of Leishmania mexicana. The antiparasitic effect was determined by incubating the parasites with different concentrations of the extract and metabolites for 5 days. Methanol extract and metabolites 1 and 2 showed remarkable antileishmanial activity (IC50 9.9 µg/ml, 2.0 and 0.9 µM, respectively) against amastigotes. These results suggested that the methanol extract of D. contrajerva and compounds 1 and 2 could serve as leads for the development of new treatments against leishmaniasis.

Plants produce compounds to resist fungal attack. However, most orchids require fungal colonisation for germination, and fully mycoheterotrophic (MH) orchids are dependent upon fungi for their entire nutritional needs. To elucidate the manner in which MH orchids interact with and accommodate a mycorrhizal partner, we examined the effect of compounds involved in pathogenic resistance on seed germination in Cyrtosia septentrionalis, an MH orchid for which a temperature of 30 °C and an atmosphere comprising reduced O2 (5%) and elevated CO2 (8%) have been shown to be optimal for axenic germination. Seeds were either co-cultured with a Physisporinus mycobiont in sealed containers or monocultured on nutrient-free water agar (WA) supplemented with various compounds under micro-aerophilic conditions (6–12% O2, 5–8% CO2) at 19, 23 or 30 °C. Co-cultured seeds germinated and underwent onward development at all temperatures. In contrast, monocultured seeds failed to germinate on WA alone except at 30 °C, but germination was initiated, as indicated by seed coat rupture, at 19 °C when K+, kinetin and NAA were added. Ca2+ and H2O2 increased germination initiation, whereas salicylic acid and gibberellic acid inhibited it. Asymbiotic initiation also occurred at 30 °C in sealed vessels with a more-or-less regular atmosphere when K+ and kinetin were added. We hypothesise that germination in C. septentrionalis is triggered by the micro-aerophilic conditions established as a result of its mycobiont’s wood-rotting activity, with certain compounds secreted as part of the early defence response having a potentiating effect. However, subsequent mycobiont colonisation is essential for full germination and onward development to ensue.

Remediation of toxic chlorinated ethenes via microbial reductive dechlorination can lead to ethene formation; however, the process stalls in acidic groundwater, leading to the accumulation of carcinogenic vinyl chloride (VC). This study explored the feasibility of cometabolic VC degradation by moderately acidophilic methanotrophs. Two novel isolates, Methylomonas sp. strain JS1 and Methylocystis sp. strain MJC1, were obtained from distinct alpine peat bogs located in South Korea. Both isolates cometabolized VC with CH4 as the primary substrate under oxic conditions at pH at or below 5.5. VC cometabolism in axenic cultures occurred in the presence (10 μM) or absence (

Bryophytes are widely used in monitoring environmental changes and various types of pollution. Features such as longevity and direct intake by the whole body make them very good models for bioindication. However, there is a considerable lack of knowledge on the coping, resistance and tolerance mechanisms to toxic elements within representatives of this second largest group of terrestrial plants. We tested two different growth form mosses, acrocarpous Atrichum undulatum and pleurocarpous Hypnum cupressiforme in axenic in vitro fully controlled laboratory conditions, thus eliminating additional side, antagonistic or synergistic effects. These mosses were exposed to three concentrations (including control) of zinc-, copper- or cadmium-acetate for a short and long period, and their growth, development and biochemical parameters were recorded. The results showed that reactions to stress are both species and metal-specific. Also, the short and long-term exposure times and the level of toxic elements played a role in species response and showed the differences between the two moss growth forms and species. Among the investigated parameters considered in the two mosses tested, the multiplication index proved to be the most useful and sensitive in detecting the metal pollution effects. On the other hand, the concentration of chl a seemed to vary over a rather small range between the stressed moss species. Overall, copper was shown to be quite toxic compared to the other two metals at lower applied concentration. However, the studied mosses demonstrated decreased viability according to all of the parameters tested in the media enriched with high concentrations of cadmium when compared with copper and zinc. These results suggest that species studies related to different metals/pollutants in small scale controlled conditions are urgently needed before application in broad areas monitored by mosses.

Attenuating the Taxol biosynthesis by fungi with storage and subculturing is the major challenge that limits their further industrial applications. Aspergillus flavipes has been reported as a potent Taxol producer, with plausible increasing to its Taxol yield upon coculturing with the microbiome of Podocarpus gracilior (El-Sayed et al., Process Biochemistry 76:55-67, 2019a; Scientific Reports 9, 2019b; Enzyme and Microbial Technology 131, 2019c); however, the identity of these microbial inducers remains ambiguous. Thus, this study was to assess the potency of individual microbes to trigger the Taxol biosynthesis by A. flavipes and to unravel the differentially expressed protein in response to bacterial interaction. Among the 25 bacterial endophytes of P. gracilior, Bacillus subtilis was the potent isolate enhancing the Taxol yield of A. flavipes by ~1.6-fold. Strikingly, this bacterial elicitor displayed a reliable inhibition to the growth of A. flavipes, so the released antifungal compound by B. subtilis could be the same signals for triggering the expression of A. flavipes Taxol synthesis. The highest Taxol yield by A. flavipes was obtained with the viable cells of B. subtilis, ensuring the pivotality of physical intimate bacterial-fungal interaction. Differential proteome of the cocultures A. flavipes and B. subtilis as well as the axenic A. flavipes was conducted by LC-MS/MS. From the total of 106 identified proteins, 50 proteins were significantly expressed, 47 were upregulated ones, and 59 were downregulated ones for the cocultures normalizing to the axenic one. From the Gene Ontology (GO) and KEGG enrichment analyses, the cellular process, primary metabolic process, and nitrogen compound metabolic process were significantly changed in the coculture normalizing to monoculture of A. flavipes. The molecular function terms (histones H2B, H2A, peptidyl-prolyl cis-trans isomerase, and nucleoside-diphosphate kinase (NDPK)) were the highly significantly expressed proteins of A. flavipes in response to B. subtilis, with strong correlation to triggering of Taxol biosynthesis. The intimate interaction of A. flavipes with B. subtilis strongly modulates the Taxol biosynthetic machinery of A. flavipes by modulating the chromatin remodeling.

Fusarium oxysporum is a plant pathogenic fungus leading to severe crop losses in agriculture every year. A sustainable way of combating this pathogen is the application of mycoparasites—fungi parasitizing other fungi. The filamentous fungus Trichoderma atroviride is such a mycoparasite that is able to antagonize phytopathogenic fungi. It is therefore frequently applied as a biological pest control agent in agriculture. Given that volatile metabolites play a crucial role in organismic interactions, the major aim of this study was to establish a method for on-line analysis of headspace microbial volatile organic compounds (MVOCs) during cultivation of different fungi. An ion mobility spectrometer with gas chromatographic pre-separation (GC-IMS) enables almost real-time information of volatile emissions with good selectivity. Here we illustrate the successful use of GC-IMS for monitoring the time- and light-dependent release of MVOCs by F. oxysporum and T. atroviride during axenic and co-cultivation. More than 50 spectral peaks were detected, which could be assigned to 14 volatile compounds with the help of parallel gas chromatography-mass spectrometric (GC-MS) measurements. The majority of identified compounds are alcohols, such as ethanol, 1-propanol, 2-methyl propanol, 2-methyl butanol, 3-methyl-1-butanol and 1-octen-3-ol. In addition to four ketones, namely acetone, 2-pentanone, 2-heptanone, 3-octanone, and 2-octanone; two esters, ethyl acetate and 1-butanol-3-methylacetate; and one aldehyde, 3-methyl butanal, showed characteristic profiles during cultivation depending on axenic or co-cultivation, exposure to light, and fungal species. Interestingly, 2-octanone was produced only in co-cultures of F. oxysporum and T. atroviride, but it was not detected in the headspace of their axenic cultures. The concentrations of the measured volatiles were predominantly in the low ppbv range; however, values above 100 ppbv were detected for several alcohols, including ethanol, 2-methylpropanol, 2-methyl butanol, 1- and 3-methyl butanol, and for the ketone 2-heptanone, depending on the cultivation conditions. Our results highlight that GC-IMS analysis can be used as a valuable analytical tool for identifying specific metabolite patterns for chemotaxonomic and metabolomic applications in near-to-real time and hence easily monitor temporal changes in volatile concentrations that take place in minutes.

To examine the impact of heterotrophic bacteria on microalgal physiology, we co-cultured the diatom Phaeodactylum tricornutum with six bacterial strains to quantify bacteria-mediated differences in algal biomass, total intracellular lipids, and for a subset, extracellular metabolite accumulation. A Marinobacter isolate significantly increased algal cell concentrations, dry biomass, and lipid content compared to axenic algal cultures. Two other bacterial strains from the Bacteroidetes order, of the genera Algoriphagus and Muricauda, significantly lowered P. tricornutum biomass, leading to overall decreased lipid accumulation. These three bacterial co-cultures (one mutualistic, two competitive) were analyzed for extracellular metabolites via untargeted liquid chromatography mass spectrometry to compare against bacteria-free cultures. Over 80% of the extracellular metabolites differentially abundant in at least one treatment were in higher concentrations in the axenic cultures, in agreement with the hypothesis that the co-cultured bacteria incorporated algal-derived organic compounds for growth. Furthermore, the extracellular metabolite profiles of the two growth-inhibiting cultures were more similar to one another than the growth-promoting co-culture, linking metabolite patterns to ecological role. Our results show that algal-bacterial interactions can influence the accumulation of intracellular lipids and extracellular metabolites, and suggest that utilization and accumulation of compounds outside the cell play a role in regulating microbial interactions.

Nitrogen is one of the most critical nutrients affecting algal cell biomass growth and thereby plays a crucial role in algal growth economics. Conventionally, all man-made nitrogen (N) sources are based on hydrogen derived from fossil fuels. This investigation examines the effect of a new N source, i.e., plasma-activated water (PAW), on yields of algal biomass. The N uptake rates of the algae for PAW were determined and it was compared with the conventional N sources. Four treatments of N source in duplicates were run to evaluate the potential of algae cultivation in batch microcosms (5 L). Mixotrophic algal cultures from a nearby eutrophied lake were incubated for 1 week with nitrate-N (NaNO3), nitrite-N (NaNO2), nitrate+nitrite (NaNO3+NaNO2), and PAW as sole N source in basal Bold’s media. Even though the cultures were not axenic, steady increase in chlorophyll and biomass was observed for all the cases studied. The highest biomass yield was observed in nitrate-N fed cultures, at an average of 32.5 mg/( $\text{L}\cdot $ day), followed by a combination of nitrate+nitrite [27.1 mg/( $\text{L}\cdot $ day)], PAW-N [26.6 mg/( $\text{L}\cdot $ day)], and nitrite-N [22.5 mg/( $\text{L}\cdot $ day)] fed cultures. Nitrogen uptake rates were similar in all cultures studied. The highest rates observed were in nitrite-N [4.05 mg/( $\text{L}\cdot $ day)], closely followed by PAW [3.56 mg/( $\text{L}\cdot $ day)]. The results suggest that PAW can be utilized as a N source with pre-exposed cultures. The algal biomass yields of PAW were comparable to conventional sources although cultures with PAW showed signs of better uptake of N.

Synechococcus accounts for a considerable proportion of oceanic carbon fixation and has close relation with heterotrophic bacteria at sub-micrometre scales. However, the relationship between Synechococcus and heterotrophic bacteria and their impacts on oceanic carbon flow is not well understood. Here, we separately co-cultured the heterotrophic bacterial community with two different axenic Synechococcus strains, i.e. Synechococcus sp. PCC7002 and Synechococcus sp. CCMP1334. The dissolved organic carbon (DOC) released by Synechococcus promoted the rapid growth of heterotrophic bacteria. Due in part to the slight differences in the Synechococcus-derived DOC composition, the starting heterotrophic bacterial community was reshaped by the two Synechococcus strains into significantly divergent community structures, suggesting that different heterotrophic bacterial communities may gather around different Synechococcus strains and interact with each other in the oceans. Interestingly, the two Synechococcus strains also released some very stable humic-like DOC components that resisted use by heterotrophic bacteria and were gradually accumulated in seawater, implying that in addition to photosynthetic carbon fixation, Synechococcus may also play an important role in long-term ocean carbon sequestration by producing relatively recalcitrant DOC. Moreover, the interactions of Synechococcus and the heterotrophic bacterial communities could increase aggregate formation and particle sinking, which can likely enhance the contribution of Synechococcus to the magnitude of the sinking biological carbon pump in the oceans.

This study describes in vitro propagation and cryopreservation of Hovenia dulcis, a woody species used in traditional medicine. Stem and leaf explants from axenic seedlings were cultivated on Murashige and Skoog (MS) medium containing 6-benzyladenine (BA) and kinetin (KIN) alone or in combination (0.1, 0.2, 0.5 mg L−1). For in vitro propagation, rates of regeneration (percentage of responsive explants) and proliferation (multiplication capacity of explant-derived shoots) were evaluated after 30 days and five subcultures, respectively. For cryopreservation by V Cryo-plate technique, shoot tips were excised from microcuttings cultured from in vitro-grown stock plants, or excised directly from axillary shoots of stock plants. The shoot tips were precultured in 0.3 M sucrose (24 h), exposed to loading (20 min) and to PVS2 (0–150 min) before storage in liquid nitrogen. The regrowth was assessed by plating of shoot tips on recovery medium (MS with BA + KIN), with or without a sterile filter paper over the culture medium. Cryopreservation was evaluated by survival (4-weeks) and recovery (8-weeks). The highest regeneration by direct organogenesis (100%) were reached on medium with BA + KIN (0.5 mg L−1 each). Shoots maintained multiplication capacity, showing the highest proliferation (87%) in the presence of BA. Shoot elongation and rooting were achieved on growth regulator-free MS. The most efficient cryopreservation protocol (68% survival and 62% recovery) applied exposure to PVS2 (120 min), and recovery on medium containing BA + KIN (0.5 mg L−1 each) with filter paper. The propagation and cryopreservation of H. dulcis may contribute to its conservation and that of other woody species. This study aimed to establish an in vitro propagation methodology and the first cryopreservation protocol for Hovenia dulcis, a woody species of commercial, medicinal and nutraceutical values.

In this study, we investigated oxidative stress in the green microalgae, Chlorella sorokiniana, in co-culture with the plant growth promoting bacteria (PGPB), Azospirillum brasilense. This relationship was studied in the absence of an exogenous stressor, under copper stress, and under nitrogen limitation stress. We confirmed that copper and nitrogen limitation induced algal oxidative stress and reductions in chlorophyll content. In all cases, the presence of A. brasilense lowered the accumulation of intracellular reactive oxygen species (ROS) while promoting chlorophyll content. This effect was driven, in part, by A. brasilense's secretion of the auxin hormone, indole-3-acetic acid, which is known to mitigate stress in higher plants. The findings of the present study show that stress mitigation by A. brasilense resulted in suppressed starch accumulation under nitrogen limitation stress and neutral lipid accumulation under copper stress. In fact, A. brasilense could almost completely mitigate oxidative stress in C. sorokiniana resulting from nitrogen limitation, with ROS accumulation rates comparable to the axenic control cultures. The biotechnological implication of these findings is that co-culture strategies with A. brasilense (and similar PGPB) are most effective for high growth applications. A second growth stage may be needed to induce accumulation of desired products.

Camptothecin the third most in demand alkaloid, is commercially extracted in India from the endangered plant, Nothapodytes nimmoniana. Endophytes, the microorganisms that reside within plants, are reported to have the ability to produce host–plant associated metabolites. Hence, our research aims to establish a sustainable and high camptothecin yielding endophyte, as an alternative source for commercial production of camptothecin. A total of 132 endophytic fungal strains were isolated from different plant parts (leaf, petiole, stem and bark) of N. nimmoniana, out of which 94 were found to produce camptothecin in suspension culture. Alternaria alstroemeriae (NCIM1408) and Alternaria burnsii (NCIM1409) demonstrated camptothecin yields up to 426.7 ± 33.6 µg/g DW and 403.3 ± 41.6 µg/g DW, respectively, the highest reported production to date. Unlike the reported product yield attenuation in endophytes with subculture in axenic state, Alternaria burnsii NCIM1409 could retain and sustain the production of camptothecin up to ~ 200 μg/g even after 12 continuous subculture cycles. The camptothecin biosynthesis in Alternaria burnsii NCIM1409 was confirmed using 13C carbon labelling (and cytotoxicity analysis on different cancer cell lines) and this strain can now be used to develop a sustainable bioprocess for in vitro production of camptothecin as an alternative to plant extraction.

Microbioassays allow for efficient contamination monitoring and control strategies. Free-living microalgae, representative of the aquatic environment, are the most used organisms due to high sensitivity and reproducibility. However, a lack of testing methods representative of terrestrial habitats has long been highlighted. A good unexploited option is the use of lichen phycobionts. The use of appropriate biomarkers leads to a reduction in costs and number of organisms, contributing to cost-efficient, rapid, and sensitive microbioassays. With the aim to develop a fast microbioassay, axenic Asterochloris erici was grown on treated cellulose paper, desiccated and rehydrated with different concentrations of inorganic and organic pollutants. Chlorophyll autofluorescence and free radical content were measured 5 min post-rehydration as energetics and oxidative status biomarkers respectively. Fluorescence microscopical images of exposed phycobionts were also collected. Potassium dichromate and copper sulphate decreased chlorophyll autofluorescence at high concentrations whereas boric and clofibric acids had little effect, all showing LOECs similar to those found in the literature. Heavy metals induced free radical bursts at extremely low concentrations whereas boric and clofibric acid showed modest and fluctuant increases. Microscopical images support fluorometric results and relate free radical bursts with bigger cells. In every case, free radicals LOEC is lower than chlorophyll autofluorescence’s by at least three orders of magnitude, making this microbioassay highly sensitive and fast, as well as low cost and ecologically relevant.

The cultivation of cyanobacteria with the addition of an organic carbon source (meaning as heterotrophic or mixotrophic cultivation) is a promising technique to increase their slow growth rate. However, most cyanobacteria cultures are infected by non-separable heterotrophic bacteria. While their contribution to the biomass is rather insignificant in a phototrophic cultivation, problems may arise in heterotrophic and mixotrophic mode. Heterotrophic bacteria can potentially utilize carbohydrates quickly, thus preventing any benefit for the cyanobacteria. In order to estimate the advantage of the supplementation of a carbon source, it is essential to quantify the proportion of cyanobacteria and heterotrophic bacteria in the resulting biomass. In this work, the use of quantitative polymerase chain reaction (qPCR) is proposed. To prepare the samples, a DNA extraction method for cyanobacteria was improved to provide reproducible and robust results for the group of terrestrial cyanobacteria. Two pairs of primers were used, which bind either to the 16S rRNA gene of all cyanobacteria or all bacteria including cyanobacteria. This allows a determination of the proportion of cyanobacteria in the biomass. The method was established with the two terrestrial cyanobacteria Trichocoleus sociatus SAG 26.92 and Nostoc muscorum SAG B-1453-12a. As proof of concept, a heterotrophic cultivation with T. sociatus with glucose was performed. After 2 days of cultivation, a reduction of the biomass partition of the cyanobacterium to 90% was detected. Afterwards, the proportion increased again.

Umbilicaria muhlenbergii is the only known dimorphic lichenized fungus that grows in the hyphal form in lichen thalli but as yeast cells in axenic cultures. However, the regulation of yeast-to-hypha transition and its relationship to the establishment of symbiosis are not clear. In this study, we show that nutrient limitation and hyperosmotic stress trigger the dimorphic change in U. muhlenbergii. Contact with algal cells of its photobiont Trebouxia jamesii induced pseudohyphal growth. Treatments with the cAMP diphosphoesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) induced pseudohyphal/hyphal growth and resulted in the differentiation of heavily melanized, lichen cortex-like structures in culture, indicating the role of cAMP signaling in regulating dimorphism. To confirm this observation, we identified and characterized two Gα subunits UmGPA2 and UmGPA3. Whereas deletion of UmGPA2 had only a minor effect on pseudohyphal growth, the ΔUmgpa3 mutant was defective in yeast-to-pseudohypha transition induced by hyperosmotic stress or T. jamesii cells. IBMX treatment suppressed the defect of ΔUmgpa3 in pseudohyphal growth. Transformants expressing the UmGPA3G45V or UmGPA3Q208L dominant active allele were enhanced in the yeast-to-pseudohypha transition and developed pseudohyphae under conditions noninducible to the wild type. Interestingly, T. jamesii cells in close contact with pseudohyphae of UmGPA3G45V and UmGPA3Q208L transformants often collapsed and died after coincubation for over 72 h, indicating that improperly regulated pseudohyphal growth due to dominant active mutations may disrupt the initial establishment of symbiotic interaction between the photobiont and mycobiont. Taken together, these results show that the cAMP-PKA pathway plays a critical role in regulating dimorphism and symbiosis in U. muhlenbergii.

The green alga Chlorella vulgaris has potential as a source of clean bioenergy with abundant metabolites and a high oil content, and antibiotics are often applied to remove bacteria from culture to obtain axenic algal strains. In this work, ceftazidime and gentamicin sulphate (GS) were added separately and in combination in the aseptic processing of C. vulgaris, and gene expression and metabolites were evaluated. The results showed that ceftazidime and GS effectively inhibited the proliferation of Cyanobacteria and Bacteroidetes, respectively. Overall, the effects of antibiotics on C. vulgaris differed: GS increased the algal concentration, whereas ceftazidime alone and in combination with GS treatment decreased the specific algal growth rate. Based on comparative transcription analysis, 5917 and 5899 differentially expressed genes (DEGs) were respectively upregulated and downregulated by ceftazidime, 963 and 3921 DEGs by GS, and 4532 and 1675 DEGs by the ceftazidime and GS combination. Pathway enrichment analysis showed that the downregulated DEGs in the ceftazidime groups were enriched in the fatty acid biosynthesis pathway but that the upregulated DEGs in the GS group were enriched in the fatty acid degradation pathway. Some pathways related to amino acid metabolism were markably influenced by antibiotic treatment. The results further indicated that antibiotics affected the intracellular concentration of fatty acids and amino acids in C. vulgaris. This study provides a new viewpoint regarding the response of C. vulgaris to antibiotics in the process of obtaining axenic algal strains. KEY POINTS: • Ceftazidime and gentamicin sulphate influenced bacterial proliferation. • Downregulated differentially expressed genes mapped to the fatty acid biosynthesis pathway. • Antibiotics affected intracellular concentrations of fatty acids and amino acids.

Fungi are a rich source of bioactive compounds. Fungal cocultivation is a method of potentiating chemical interactions and, consequently, increasing bioactive molecule production. In this study, we evaluated the bactericidal, antiprotozoal, and cathepsin V inhibition activities of extracts from axenic cultures of 6 fungi (Fusarium guttiforme, Pestalotiopsis diospyri, Phoma caricae-papayae, Colletotrichum horii, Phytophthora palmivora, and C. gloeosporioides) that infest tropical fruits and 57 extracts obtained by their cocultivation. Our results reveal that fungal cocultivation enhances the biological activity of the samples, since all extracts that were active on Gram-positive bacteria, Gram-negative bacteria, Trypanosoma cruzi, and Leishmania infantum were obtained from cocultivation. Bacterial growth is either totally or partially inhibited by 46% of the extracts. Two extracts containing mainly fusaric and 9,10-dehydrofusaric acids were particularly active. The presence of the fungus F. guttiforme in co-cultures that give rise to extracts with the highest activities against L. infantum. An axenic culture gave rise to the most active extract for the inhibition of cathepsin V; however, other coculture extracts also exhibited activity toward this biological target. Therefore, the results of the biological activities indicate that fungal cocultivation increased the biological potential of samples, likely due to the hostile and competitive environment that pushes microorganisms to produce substances important for defense and allows access to metabolic routes then silenced in milder cultivation conditions.

Dissolved organic carbon (DOC) is an important source of carbon and energy for microbes, but whether it can be used by phytoplankton has not been systematically studied, and the underlying molecular metabolism remains poorly understood. Here, we investigated the ability of phytoplankton to utilize glycerol as a representative of DOC. The widespread presence and expression of glycerol transporter genes were found in transcriptomes and genomes, suggesting the glycerol utilization potential in the diverse marine phytoplankton. We surveyed 29 representative phytoplankton species (31 strains) from six phyla for their ability to use glycerol. Three types of responses were found: positive utilization (Type I), no response (Type II), and negative response (Type III). In all, 11 Type I species were further investigated in axenic cultures with different glycerol concentrations, and five species showed intrinsic glycerol utilizing ability without the aid of bacteria. The ability of species to use glycerol to support non-photosynthetic (DCMU treated) growth was consistent with their possession and expression of glycerol transporter genes. However, some species from the Type II and Type III also possess and express the genes, raising a question whether glycerol transporter in algae might have diversified its function to glycerol export or even non-glycerol transport. Our results show that glycerol could serve as organic carbon source, harmless xenobiotics, or growth inhibitors for phytoplankton depending on species, indicating that glycerol, including natural sources or human discharges, may shape the marine phytoplankton community structure, especially under low photosynthetic efficiency conditions.

Light mediates plant growth through diverse mechanisms and signaling networks including plant growth regulators (PGRs). We hypothesized that a novel class of PGRs, the indoleamines, are plant signaling molecules that perceive changes in light composition and initiate a cascade of metabolic responses. We used three Scutellaria model species (skullcap): S. lateriflora, S. galericulata and S. racemosa that produce high levels of melatonin and serotonin to investigate this hypothesis. Axenic Scutellaria cultures were exposed to red, blue, green or full spectrum white light spectra provided by light emitting diode (LED) lighting systems, or daylight fluorescent bulbs. Melatonin (MEL), serotonin (5HT), abscisic acid (ABA), auxin (IAA), and jasmonic acid (JA), were quantified by liquid chromatography with tandem mass spectrometry. Melatonin was detected consistently in plants grown under blue light in all species of Scutellaria. In S. galericulata, significant quantities of ABA were detected in plants grown under white light but not detected in plants grown under other light spectra. In timeline studies of S. racemosa plants exposed to limited red or blue light spectra had significantly reduced levels of tryptamine (TRM), 5HT and MEL in the shoots initially but melatonin was detected after 12 hours and quantifiable amounts of 5HT were detected after 7 days. Supplementation of the culture medium with MEL or 5HT did not change the pattern of MEL in blue light grown cultures but did change patterns of 5HT accumulation. 5HT was highest in plants grown under red light immediately after culture and decreased over 7 days. These data indicate that the relative amounts of MEL and 5HT are responsive to light spectra and redirect metabolic resources to enable plant adaptations to changing environments.

Rhodiola rosea L. is an endangered medicinal plant distributed in mountains and in high latitude regions. For its conservation, sustainable methods for the obtaining of its bioactive compounds must be developed. This work hypothesized that leaf, stem and rhizome explants of R. rosea from different geographical origins respond differently to inoculation with Rhizobium rhizogenes agropine strain ATCC43057. The objective was to generate R. rosea hairy roots (HRs) containing rol-genes. These HRs could be cultivated under axenic conditions for the extraction of the medical compounds rosavinoids and salidroside. Hereby, production of bioactive compounds could be improved per plant biomass. Thirteen R. rosea accessions of Alpine, Scandinavian, Nordic Gene Bank (NGB) and Russian origins were compared for their explant survival and HR formation. Significant differences were observed among plants from different geographical origins, where the NGB leaf explants exhibited up to 70% of HR formation and the Russian accessions did not exhibit HRs at all. Moreover, maintaining explants in light conditions after R. rhizogenes inoculation resulted in higher explant survival and HR formation rate (35%) when compared with explants kept in darkness (9%). Taken together, an efficient HR formation in roseroot by inoculation of R. rhizogenes following culturing in light was reported as a required step. This work represents a stepping-stone to R. rosea HR cultivation in bioreactors as well as regenerating whole plants. Hence, it is initiating a novel route towards high-throughput production of bioactive compounds as well preventing depletion of natural roseroot populations.

This study was designed to assess the synergistic effects of nitrogen (N) and phosphorus (P) concentrations on oil content, fatty acid profile, and predicted fuel properties of Dunaliella salina. Axenic D. salina cells were grown in F/2 growth medium of salinity 34 ppt containing 33.6 g.l−1 ultramarine synthetic sea salt. Growth dry weight, cell count, and their relationship were measured, and oils were extracted by soaking following Soxhlet extraction. Growth dry weight was markedly affected by N and P concentrations, with maximum growth dry weights of cultures grown at recommended N and P concentrations (control), half of the recommended N concentration (0.5 N) and (0.5 N/0.5P) being 0.911 g.l−1, 0.755 g.l−1, and 0.615 g.l−1, respectively. Oil content showed the reverse pattern, with cultures grown in the absence of phosphorus (0.0P), full N/P starvation (0.0 N/0.0P), and control resulting in maximum oil contents of 24.86%, 22.85%, and 5.88%, respectively. The majority of fatty acid methyl esters ranged between C14 and C22. Estimated fuel properties of algal cells grown under NP stress conditions were found to meet the American Society for Testing and Materials (ASTM) and European Committee for Standardization (EN) guidelines.

Co-cultivation of viable cells of the human pathogenic bacterium Pseudomonas aeruginosa with the endophytic fungus Fusarium tricinctum induced silent bacterial biosynthetic gene clusters. Visual and chemical inspection of the co-cultures by comparison with the axenic cultures showed a clear reaction of P. aeruginosa towards its fungal competitor which was reflected by the deep greenish coloration of the co-cultures. Inspection of the HPLC chromatograms of the co-cultures revealed the induction of the pseudomonal quorum sensing molecule 2-heptyl-4-hydroxy-quinolone (HHQ) along with the antifungal phenazine alkaloids, phenazine-1-carboxylic acid (PCA) and phenazine-1-carboxamide (PCN), which are well-known metabolites of P. aeruginosa. The latter compounds were not detected in axenic bacterial cultures. In contrast to P. aeruginosa, no metabolic responses were detected for F. tricinctum which is in sharp contrast to previous co-culture experiments with this fungus using other bacteria.

Free-living amoebae (FLA) are widely dispersed in the environment, can cause opportunistic and non-opportunistic infections in humans and other animals. The aim of the present study was characterize FLA obtained from air-conditioners of a public hospital in the city of Florianópolis, SC, Brazil. Fifty-four dust samples were collected of air conditioners, and were inoculated on 1.5% non-nutrient agar, overlaid with layers of Escherichia coli. Subsequently the isolates were axenised in PYG growth medium. The morphological and molecular characterization of the isolates was performed, as well as the tolerance (physiological) assays were used to evaluate the pathogenic potential. The results revealed the presence of FLA in 42 (77.8%) of the collected samples. Of these, 39 (92.9%) axenic isolates of FLA were obtained for morphological and genotypic studies. All the isolates characterized belong to the genus Acanthamoeba. Nineteen (48.7%) isolates belong to the genotype T4, 16 (41.0%) to the T5 genotype and 4 (10.3%) to genotype T11. Seven (18.0%) isolates were considered potentially pathogenic in tolerance assays. These findings require attention, considering the isolation environment and immunocompromised characteristics of many hospitalized patients.

In vitro mitotic polyploidization using anti-microtubule agents has been commonly used for polyploid production. The present study was an attempt to develop an in vitro oryzalin chromosome doubling and stable tetraploid regenerating protocol for Dendrobium officinale. In this study, we successfully induced tetraploids by treating protocorms developed from the seeds of multiple genotypes. The highest frequency of polyploidy was 37.40%, achieved with 14.4 µM oryzalin treatment for 24 h. Among the obtained polyploid seedlings, 72 solid tetraploid, 33 mixoploid and 3 octoploid genotypes were identified via screening using flow cytometry (FCM). Three peaks were observed in the histograms of the diploid, tetraploid, and octoploid leaves, but four peaks were observed only in mixoploid leaves with FCM, indicating the existence of endopolyploid cells and the occurrence of conventional endoreduplication in the leaves of all ploidy levels. Recurrent ploidy identification of various tetraploid genotype regenerated plantlets obtained via protocorm-like bodies (PLBs) derived from axenic stem-node segments maintained stable tetraploid levels. Comparisons of phenotypic characteristics revealed that relative to the diploid plantlets, the tetraploid plantlets exhibited increased stem diameter, root diameter, labellum width and gynostemium width. Furthermore, the tetraploid plantlets showed lower plant height, leaf length and root length than the diploid plantlets. This efficient polyploid induction and ploidy-stable regeneration protocol can be used for the mass production of tetraploid D. officinale. The tetraploid genotypes regenerated in this study might be useful for nobile Dendrobium breeding in the future. In vitro induction of various tetraploid genotypes in D. officinale and subsequent rapid micropropagation through induction of PLBs from axenic nodal segments were performed to obtain ploidy-stable regenerated plantlets

As a consequence of axenic growth and the elimination of accompanying bacterial flora,Entamoeba histolyticavirulence decreases rapidly, and pathogenicity is lost. This paper evaluated the impact of vitamin supplementation on the pathogenicity ofE. histolytica.Growth ofE. histolyticatrophozoites, cultured axenically in PEHPS (a Spanish acronym for the main ingredients – casein peptone, liver, pancreas extract and bovine serum) medium, with or without vitamins, exhibited a similar growth rate. However, the vitamin-enriched PEHPS preparations expressed 2.65 times more haemolytic activity (at 60 min: 98vs48%,P< 0.05), 2.5 times more phospholipase A2activity at 150 min of incubation and generated more hepatic abscesses (88vs60%,P= 0.05) than the preparations without vitamins. The haemolytic and phospholipase A2activity for the PEHPS − V preparations were restored following vitamin supplementation with A and D. These data highlight, for the first time, that vitamins and specifically vitamin A and D were essential for the recovery of amoebic virulence, lost through axenic growth.

Cobalt oxide nanoparticles were successfully biosynthesized by complete green process using aqueous leaf extracts of Sageretia thea as chelating agent. Diverse techniques were applied for characterization. Antibacterial (with and without UV illumination), antileishmanial, antioxidant and enzyme inhibition applications were assessed, while freshly isolated macrophages and red blood cells were used for biocompatibility studies. Good antibacterial nature and enhancement of bactericidal nature upon UV modulation is reported. Staphylococcus aureus and Escherichia coli are indicated as most susceptible bacterial strains. Significant cytotoxic potential is revealed with IC50 calculated as 12.82 µg/ml and 3.16 µg/ml against the axenic leishmanial promastigote and amastigote cultures respectively. Biogenic cobalt oxide nanoparticles indicated DPPH free radical scavenging potential, while moderate antioxidant capacity and reducing power was demonstrated. Bioinspired cobalt oxide also demonstrated alpha amylase and protein kinase inhibition at higher concentrations. Biogenic cobalt oxide was found as more cytotoxic to macrophages (IC50 = 58.55 µg/ml) then to RBC’s (IC50 >200 µg/ml). Our results indicate green synthesis as an alternative, effective and eco-friendly method for the biosynthesis of cobalt oxide nanoparticles with numerous biological applications. Keywords: Biosynthesis, Cobalt oxide, Nanoparticles, Antimicrobial, Antileishmanial, Antioxidant

Sugarcane straw (SS) was used in an economic biosystem to evaluate the production of xylanases and cellulases in submerged fermentation (SmF) by axenic and mixed mode from Trichoderma and Aspergillus species. T. reesei QM9414 axenic culture reached the highest xylanase production (90.2 U/mL) and 0.5 FPU/mL of cellulase activity. The evaluation of agro-industrial residues on fibrolytic enzymes production was performed by a D-optimal design, and revealed the best supplementation of 100% SS, while wheat bran and citric pulp showed lower inductive effects on enzymes production. Also, the scale-up in a stirred tank showed the same yield production profile (xylanase ~ 90 U/mL and celullase 0.6 FPU/mL). Xylanase was characterized by an optimum pH of 5-6 and temperature at 50 oC, and thermal stability was below 50 oC. The ion Mn2+ (5 and 10 mM) had a stimulatory effect on xylanase activity. The biobleaching application showed that 30 U/g of xylanases during 15 min decreased Kappa number in 9.37. These results indicate SS as an alternative substrate for fungi fibrolytic enzymes production and the xylanase with low cellulase extract as a potential biobleaching application.

Manganese (Mn) is an essential micronutrient for plant growth that is involved in the structure of photosynthetic proteins and enzymes. Mn deficiency is widespread mainly in dry, calcareous, and sandy soil, which leads to a significant decrease in crop yield. Mn-reducing bacteria promote the solubilization of Mn minerals, thus increasing Mn availability in soil. The present study aimed to assess the Mn solubilizing ability and plant growth-promoting potential of Bacillus spp. strains for maize plants with insoluble Mn compounds. Several Mn-solubilizing bacterial (MSB) strains were isolated from the maize rhizosphere using nutrient agar media amended with 50 mM MnO2. These strains were screened based on qualitative and quantitative solubilization of Mn, phosphorus, potassium, and zinc and production of ammonia. The majority of MSB strains were positive for catalase, protease, amylase, and oxidase activity, while more than 60% of tested strains were positive for lipase activity, and the production of indole-3-acetic acid and siderophores. Forty-five percent of the tested strains also showed solubilization of potassium. All the MSB strains were evaluated for their ability to promote plant growth and Mn uptake in the presence of MnO2 under axenic sand culture conditions. The results revealed that inoculation with MSB strains under sand culture significantly improved the growth of maize seedlings except for strains ASH7, ASH10, and ASH12. Comparatively, strains ASH6, ASH11, ASH19, ASH20, and ASH22 demonstrated a better increase in plant growth, fresh and dry biomass, and Mn uptake in roots and shoots than the other strains tested. All of these strains were identified as Bacillus spp. through 16S rRNA partial gene sequencing. Maize inoculation with these selected identified MSB strains also resulted in an increase in maize growth and nutrient uptake in maize roots and shoots under soil culture conditions in the presence of native soil Mn. The current study highlights the importance of MSB strain inoculation which could be a potential bioinoculants to promote plant growth under Mn deficiency.

Axenic fermentation on solid rice of the saprobic fungus Sparticola junci afforded two new highly oxidized naphthalenoid polyketide derivatives, sparticatechol A (1) and sparticolin H (2) along with sparticolin A (3). The structures of 1 and 2 were elucidated on the basis of their NMR and HR-ESIMS spectroscopic data. Assignment of absolute configurations was performed using electronic circular dichroism (ECD) experiments and Time-Dependent Density Functional Theory (TDDFT) calculations. Compounds 1–3 were evaluated for COX inhibitory, antiproliferative, cytotoxic and antimicrobial activities. Compounds 1 and 2 exhibited strong inhibitory activities against COX-1 and COX-2. Molecular docking analysis of 1 conferred favorable binding against COX-2. Sparticolin H (2) and A (3) showed a moderate antiproliferative effect against myelogenous leukemia K-562 cells and weak cytotoxicity against HeLa and mouse fibroblast cells.

The microbial carbon pump (MCP) provides a mechanistic illustration of transformation of recalcitrant dissolved organic matter (DOM) in the ocean. Here, we explored and demonstrated the key roles of algae-associated microorganisms (mainly heterotrophic bacteria) in the production and transformation of carboxyl-rich alicyclic molecule (CRAM)-like DOM through a laboratory experiment involving cultures of Skeletonema dohrnii. Without the participation of the associated bacteria, CRAM-like DOM molecules were not detected via Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) in algal cultures treated with antibiotics. Similarly, CRAM-like DOM were not detected in cultures of bacteria alone. Our experimental results showed that algae-associated bacteria are important in the process of converting algal-derived organic matter into CRAM-like DOM during S. dohrnii culture. Bacteroidetes (mainly Flavobacteriia) dominated the bacterial community in the stationary and degradation phases, where the predicted metabolic pathways for bacterial assemblages were mainly involved in biosynthesis, metabolism, and degradation. Facilitated by these heterotrophic bacteria, the amount and the chemodiversity of CRAM-like DOM derived from algae varied during the growth and decomposition of algal cells, and CRAM-like DOM were enriched at the later growth stage. The properties and characteristics of these CRAM-like DOM, including molecular weight, double bond equivalent, hydrogen-carbon ratio, carbon-nitrogen ratio, carbon-sulfur ratio, and modified aromaticity index increased with the growth and decay of algal cells, indicating the transformation from active to recalcitrant DOM. In contrast, the organic matter in axenic cultures of S. dohrnii mainly existed in the form of particulate organic matters (POM), and small amounts of CRAM-like DOM were detected. This study provides the first laboratory evidence to reveal and confirm the direct involvement of algae-associated microbiomes in the production and transformation of algae-derived refractory DOM, highlighting the significance of these epiphytic bacteria in marine carbon sequestration and global carbon cycling. IMPORTANCE Dissolved organic matter (DOM) serves as a major carbon and nutrient pool in oceans, and recalcitrant DOM are the primary sources for carbon sequestration in depths. Here, we demonstrate the critical roles of algae-associated microorganisms (mainly heterotrophic bacteria) in the transformation of recalcitrant dissolved organic matter through laboratory cultures of a model diatom, Skeletonema dohrnii. Our experimental results showed that in addition to affecting the growth and the physiology of S. dohrnii, algae-associated bacteria are important in processing and converting algal DOM into CRAM-like DOM. Facilitated by the associated bacteria, the amount and the chemodiversity of DOM derived from algae varied during the growth and decomposition of algal cells, and enriched recalcitrant DOM formed in the later growth stage. The properties and diversity of DOM increased with the growth and decay of algal cells, indicating the transformation from active DOM to inert organic matter. Our results confirmed that the direct involvement of algae-associated microbes in the production of CRAM-like DOM. Detailed community structure analysis of the algae-associated bacterial community and its predicted functions confirmed the involvement of certain bacterial groups (e.g., Flavobacteriia) in biosynthesis, metabolism, and degradation.

Heavy metal and petroleum contaminated sites comprise a mixed culture of inherent microbial species yet the occurrence of competition for nutrients causes interference within species cooperating the biodegradation. Insights into the biodiversity of the contaminated sites and extreme conditions of oil reservoirs decipher the presence of both hydrocarbon degraders and biosurfactant-producing microbes. Biosurfactant-producing strains improves the bioavailability of hydrocarbons by emulsifying them or solubilize them using micellar structure formation, making it easy for the hydrocarbon degraders to metabolize them. Such inherent microbes can be designed into a stable microbial consortium harboring resilience, division of labor and resistance to extreme environmental drifts in order to improve the overall biodegradation efficiency. A top-down approach involves selectively enriching the active degraders within the inherent microbes. Yet, non-cultivable microbes are neglected in this approach reducing the overall biodegradation efficiency. Another bottom-up approach allows the inclusion of engineered microbes with equipped enzymes with the indigenous microbes for improving the overall consortium performance. Studies support the suitability of mutualism and commensalism as key interactions for consortium stability. Various phenomena such as syntrophy, biotransformation, detoxification and biofilm formation are the result of such interaction within a consortium. However, the culture conditions for these mutually interactive species need to be further optimized to improve the overall biodegradation and biosurfactant production. This chapter reports the various micro-consortium designs prepared to improve the overall biodegradation efficiency over axenic cultures.

Environmental pollution by non-biodegradable polyethylene (PE) plastics is of major concern, thus, organisms capable of bio-degrading PE are required. The larvae of the Greater Wax Moth, Galleria mellonella (Gm), were identified as a potential candidate to digest PE. In this study, we tested whether PE was metabolized by Gm larvae and could found in their tissues. We examined the implication of the larval gut microbiota by using conventional and axenic reared insects. First, our study showed that neither beeswax nor PE alone favour the growth of young larvae. We then used Fourier-Transform Infrared Microspectroscopy (µFTIR) to detect deuterium in larvae fed with isotopically labelled food. Perdeuterated molecules were found in most tissues of larvae fed with deuterium labelled oil for 72 hours proving that µFTIR can detect metabolization of 1-2 mg of deuterated food. No bio-assimilation was detected in the tissues of larvae fed with 1-5 mg of perdeuterated PED4 for 72 hours and 19-21 days, but micron sized PE particles were found in the larval digestive tract cavities. We evidenced weak bio-degradation of PE films in contact for 24 hours with the dissected gut of conventional larvae; and in the PED4 particles from excreted larval frass. Our study confirms that Gm larvae can bio-degrade PE but can not necessarily metabolize it.

Fusarium graminearum species complex produces type B trichothecenes oxygenated at C-7. In axenic liquid culture, F. graminearum mainly accumulates one of the three types of trichothecenes, namely 3-acetyldeoxyinvalenol, 15-acetyldeoxyinvalenol, or mixtures of 4,15-diacetylnivalenol/4-acetylnivalenol, depending on each strain’s genetic background. The acetyl groups of these trichothecenes are slowly deacetylated to give deoxynivalenol (DON) or nivalenol (NIV) on solid medium culture. Due to the evolution of F. graminearum FgTri1, encoding a cytochrome P450 monooxygenase responsible for hydroxylation at both C-7 and C-8, new derivatives of DON, designated as NX-type trichothecenes, have recently emerged. To assess the risks of emergence of new NX-type trichothecenes, we examined the effects of replacing FgTri1 in the three chemotypes with FgTri1_NX chemotype, which encodes a cytochrome P450 monooxygenase that can only hydroxylate C-7 of trichothecenes. Similar to the transgenic DON chemotypes, the transgenic NIV chemotype strain accumulated NX-type 4-deoxytrichothecenes in axenic liquid culture. C-4 oxygenated trichothecenes were marginal, despite the presence of a functional FgTri13 encoding a C-4 hydroxylase. At present, outcrossing of the currently occurring NX chemotype with NIV chemotype strains of F. graminearum in the natural environment likely will not yield a new strain that produces a C-4 oxygenated NX-type trichothecene.

In preparation for whole-genome sequencing, the axenic culture for two strains of a freshwater diatom Fragilaria radians were produced. Although their axenicity was controlled for the cultures’ entire lifetime, the published genomic assembly was later found to contain a large amount of bacterial sequences. Using various in silico analyses of whole genome read libraries and 16S rRNA sequencing of culture samples, we reconstruct the history of the contamination and document the failures of various axenicity control methods. This knowledge is used to discuss how these failures could have been avoided, and to provide guidelines for future works on axenic diatom cultures.

Numerous diverse microorganisms reside in the cold desert soils of continental Antarctica, though we lack a holistic understanding of the metabolic processes that sustain them. Here, we profile the composition, capabilities, and activities of the microbial communities in 16 physicochemically diverse mountainous and glacial soils. We assembled 451 metagenome-assembled genomes from 18 microbial phyla and inferred through Bayesian divergence analysis that the dominant lineages present are likely native to Antarctica. In support of earlier findings, metagenomic analysis revealed that the most abundant and prevalent microorganisms are metabolically versatile aerobes that use atmospheric hydrogen to support aerobic respiration and sometimes carbon fixation. Surprisingly, however, hydrogen oxidation in this region was catalyzed primarily by a phylogenetically and structurally distinct enzyme, the group 1l [NiFe]-hydrogenase, encoded by nine bacterial phyla. Through gas chromatography, we provide evidence that both Antarctic soil communities and an axenic Bacteroidota isolate (Hymenobacter roseosalivarius) oxidize atmospheric hydrogen using this enzyme. Based on ex situ rates at environmentally representative temperatures, hydrogen oxidation is theoretically sufficient for soil communities to meet energy requirements and, through metabolic water production, sustain hydration. Diverse carbon monoxide oxidizers and abundant methanotrophs were also active in the soils. We also recovered genomes of microorganisms capable of oxidizing edaphic inorganic nitrogen, sulfur, and iron compounds and harvesting solar energy via microbial rhodopsins and conventional photosystems. Obligately symbiotic bacteria, including Patescibacteria, Chlamydiae, and predatory Bdellovibrionota, were also present. We conclude that microbial diversity in Antarctic soils reflects the coexistence of metabolically flexible mixotrophs with metabolically constrained specialists.

The use of axenic animal models in experimental research has exponentially grown in the past few years and the most reliable way for confirming their axenic status remains unclear. It is especially the case when using individual ventilated positive-pressure cages such as the Isocage. This type of cage are at a greater risk of contamination and expose animals to a longer handling process leading to more potential stress when opened compared to isolators. The aim of this study was to propose simple ways to detect microbial contaminants with Isocages type isolator resulting by developing, validating and optimizing three different methods (culture, microscopy, and molecular). These three approaches were also tested in situ by spiking 21 axenic mice with different microorganisms. Our results suggest that the culture method can be used for feces and surface station (IBS) swabs exclusively (in Brain Heart Infusion for 7 days at 25°C and 37°C in aerobic conditions, and at 30°C in anaerobic conditions), while microscopy (wet mounts) and molecular method (quantitative PCR) were only suitable for fecal matter analyses. In situ results suggests that the culture and molecular methods can detect up to 100% of bacterial contamination events while the microscopy approach generates many erroneous results when not performed by a skilled microscopist. In situ results also suggest that when an axenic mouse is contaminated by a microbial agent, the microorganism will colonize the mouse to such an extent that detection is obvious in 4 days, in average. This report validates simple but complimentary tests that can be used for optimal detection of contaminants in axenic animal facilities using Isocage type isolators.

Cactus pear (Opuntia ficus-indica) is a crassulacean acid metabolism (CAM) species that serves as a food, feed, and bioenergy crop. O. ficus-indica is an attractive alternative biofuel feedstock due to its low water demand and high biomass productivity. Current ethanol yields from O. ficus-indica are not commercially viable due to low concentrations of released fermentable carbohydrates. Axenic strains of bacteria and fungi were isolated and characterized from a soil microbial community consortium that effectively degrades cladodes into soluble components. The consortium consisted of species representing 14 genera of eubacteria and four genera of fungi. The digestion efficiency of each axenic isolate was evaluated by measuring the release of soluble material after aerobic digestion of cladodes and direct measurement of cellulase and pectinase activities in the culture supernatants. Pectobacterium cacticida was the most effective eubacterial species identified for degrading cladodes among all isolates evaluated. Thus, P. cacticida holds great promise for increasing the release of fermentable sugars and improving overall ethanol yields., Highlights • Ethanol yields from cactus pear are not commercially viable due to poor release of fermentable carbohydrates. • A soil consortium consisting of 14 genera of eubacteria and four genera of fungi was characterized. • Pectobacterium cacticida degraded cladodes most effectively among all axenic isolates evaluated. • P. cacticida holds potential promise to promote fermentable sugar release and ethanol yields., Cactus pear, Opuntia ficus-indica, microbial consortium, cladode, fermentable sugars.

New resources of food, pharmaceuticals or biotechnological products are needed. The huge biodiversity of aero-terrestrial lichen-symbiont microalgae belonging to the Chlorophyta group remains unexplored despite they present interesting features such as extreme stress tolerance and growth in water shortage. Appropriateness for human consumption demands the demonstration of the absence of toxic effects. In vitro biocompatibility of crude homogenates of axenic microalga Asterochloris erici, isolated from the lichen Cladonia cristatella, was analyzed after treatment of cultured L929 fibroblasts with different concentrations of microalgal homogenates. The microalgal protein content (37%) was similar to spirulina or soybean. Antioxidant capacity (10.6 ± 0.6 µmol TE/g WW) or phenolic content (7.5 ± 0.5 mg GAE/g DW) were high compared to Chlorella. The results show that crude homogenates of A. erici do not induce cytotoxicity but seem to have some cytostatic effect inducing slight cell cycle alterations and intracellular reactive oxygen species (ROS) increase at the highest concentration. Carotenoid analysis demonstrates high contents of lutein (1211 µg/g microalga DW), a xanthophyll with antioxidant and cytostatic properties in vivo and high commercial added value. These findings confirm that Asterochloris erici can be suitable for the development of alimentary or pharmaceutical applications and further in vivo animal testing. The cytostatic effects should be further investigated for antitumor agents.

SUMMARYGenomic and physiological evidence from some strains of ammonia-oxidizing Thaumarchaeota demonstrate their additional ability to oxidize nitrogen (N) supplied as urea or cyanate, fueling conjecture about their ability to conserve energy by directly oxidizing reduced N from other dissolved organic nitrogen (DON) compounds. Similarly, field studies have shown rapid oxidation of polyamine-N in the ocean, but it is unclear whether Thaumarchaeota oxidize polyamine-N directly or whether heterotrophic DON remineralization is required. We tested growth of two marine Nitrosopumilus isolates on DON compounds including polyamines, amino acids, primary amines, and amides as their sole energy source. Though axenic cultures only consumed N supplied as ammonium or urea, there was rapid but inconsistent oxidation of N from the polyamine putrescine when cultures included a heterotrophic bacterium. Surprisingly, axenic cultures oxidized 15N-putrescine during growth on ammonia, suggesting co-metabolism or accelerated breakdown of putrescine by reactive metabolic byproducts. Nitric oxide, hydrogen peroxide, or peroxynitrite did not oxidize putrescine in sterile seawater. These data suggest that the N in common DON molecules is not directly accessible to marine Thaumarchaeota, with thaumarchaeal oxidation (and presumably assimilation) of DON-N requiring initial heterotrophic remineralization. However, reactive byproducts or enzymatic co-metabolism may facilitate limited thaumarchaeal DON-N oxidation.

The morphological characteristics of diatoms are useful for studying their taxonomy. However, the distinction between closely related diatom taxa can be very difficult, especially when the morphological characters are modified by environmental constraints. In the present study, 13 fresh water diatoms were identified morphologically and cultured under axenic conditions. To check this, PCR primers specific for multilocus genes were designed to amplify and screen 13 fresh water diatom monocultures. Multilocus PCR primers (DRR3, scfcpA, Lhcf11, SIT1, SIT3, SIT4, LOC101218388, COI-5P, rbcL, rbcL-3P, LSU D2/D3, UPA, psaA, and 18S rRNA) were tested. It was found that psaA gene, a plant pigment chlorophyll-based PCR marker, amplified in all the diatoms. Out of 13 diatom amplicons, only two fresh water diatoms DNA were sequenced. This included Cyclotella meneghiniana and Sellaphora pupula. The Sanger sequencing results thus established that morphologically identified diatom, Sellaphora pupula, exhibited close phylogeny to Sellaphora whereas fresh water Cyclotella meneghiniana has close lineage to marine diatom Thallosiosira.

Hyperthermophilic archaea of the genus Archaeoglobus are the subject of many fundamental and biotechnological researches. Despite their significance, the class Archaeoglobi is currently represented by only eight species obtained as axenic cultures and taxonomically characterized. Here, we report the isolation and characterization of a new species of Archaeoglobus from a deep-sea hydrothermal vent (Mid-Atlantic Ridge, TAG) for which the name Archaeoglobus neptunius sp. nov. is proposed. The type strain is SE56T (=DSM 110954T = VKM B-3474T). The cells of the novel isolate are motile irregular cocci growing at 50–85°C, pH 5.5–7.5, and NaCl concentrations of 1.5–4.5% (w/v). Strain SE56T grows lithoautotrophically with H2 as an electron donor, sulfite or thiosulfate as an electron acceptor, and CO2/HCO3− as a carbon source. It is also capable of chemoorganotrophic growth by reduction of sulfate, sulfite, or thiosulfate. The genome of the new isolate consists of a 2,115,826 bp chromosome with an overall G + C content of 46.0 mol%. The whole-genome annotation confirms the key metabolic features of the novel isolate demonstrated experimentally. Genome contains a complete set of genes involved in CO2 fixation via reductive acetyl-CoA pathway, gluconeogenesis, hydrogen and fatty acids oxidation, sulfate reduction, and flagellar motility. The phylogenomic reconstruction based on 122 conserved single-copy archaeal proteins supported by average nucleotide identity (ANI), average amino acid identity (AAI), and alignment fraction (AF) values, indicates a polyphyletic origin of the species currently included into the genus Archaeoglobus, warranting its reclassification.

Background: Glucantime® (SbV) is considered the first-line treatment against American Tegumentary Leishmaniasis in South America, though increased parasite resistance towards it have been reported and hampered its effectiveness. In this context, subtilisins serine proteases have been related to parasites’ susceptibility to drugs such as SbV and derivatives, through modulation of the parasite detoxification system. However, little is known about parasites causing ATL and their distinct responses towards this treatment.Methods: The study was conducted using Leishmania (Viannia) braziliensis clinical isolates from patients that presented clinical cure or Responders (R) and relapse/therapeutic failure or Non-responders (NR). Twelve clinical isolates were used to assess their in vitro susceptibility to SbIII and SbV, serine proteases activity, and expression of subtilisins-like and tryparedoxin-peroxidase (TXNPx) transcripts. Results: SbIII was able to better distinguish axenic amastigotes from each clinical group. These isolates were also assessed for serine protease activity, using z-FR-AMC as substrate and detecting distinct enzyme profiles with specific inhibitors. TLCK inhibited almost 100% of activity in both promastigotes and axenic amastigotes while AEBSF inhibited around 70%. PMSF showed low inhibition of specific isolates (35%). Gathering all the quantitative data, we performed principal component analysis and then used the K-means algorithm to cluster the isolates. This analysis yielded one cluster with only one isolate (R isolate), one homogeneous cluster (NR isolates), and three heterogeneous clusters (R and NR isolates). Additionally, gene transcripts of subtilisins and TXNPx were detected in promastigotes and axenic amastigotes from both groups. Conclusions: Cluster analysis showed that there is a phenotypic heterogeneity among the isolates, however, exploration of in vitro phenotypes based on SbIII and serine proteases profiles can aid in the characterization and better understanding of L. (V.) braziliensis clinical isolates.

Background Leishmania donovani, a protozoan parasite, is the primary causative agent for visceral leishmaniasis. Toxicity and increased resistance to existing drugs have led to an urgent need for identifying new drugs and drug targets. Understanding the risks and mechanisms of resistance is of great importance. Amphotericin B (AmB) is a polyene antimicrobial, the mainstay therapy for visceral leishmaniasis in several parts of India. Objectives In the present study, we established a line of AmB-resistant L. donovani promastigotes in vitro and demonstrated the molecular basis of resistance to AmB. Methods AmB-resistant promastigotes were generated and characterized to evaluate the mechanism of resistance to AmB. We examined the sterol composition of the promastigotes and the axenic amastigotes derived from the WT and AmB-resistant promastigotes. The role of the plant-like C-22 desaturase responsible for stigmasterol production was also evaluated in the AmB-resistant strain. Results The IC50 for resistant cells was four times higher than for the WT. AmB-resistant promastigotes showed an increase in the conversion of β-sitosterol into stigmasterol. The presence of higher amounts of stigmasterol in resistant promastigotes, as well as in axenic amastigotes, signifies its role in AmB resistance in Leishmania. The resistant strain showed reduced infectivity in vitro. Conclusions We have elucidated the mode of action and resistance mechanisms to the drug. However, further work is required to validate the potential role of stigmasterol in resistance and to help develop a diagnostic kit that can assist in diagnosing potentially resistant lines in the field.

The coculture of marine red algal-derived endophytic fungi Aspergillus terreus EN-539 and Paecilomyces lilacinus EN-531 induced the production of a new terrein derivative, namely asperterrein (1) and a known dihydroterrein (2), which were not detected in the axenic cultures of both strains. The production of the known secondary metabolites terrein (3), butyrolactone I (4), and dankasterone (6), derived from A. terreus EN-539, were depressed significantly in the coculture. Compounds 1–3 exhibited inhibitory activity against Alternaria brassicae, Escherichia coli, Physalospora piricola, and Staphylococcus aureus with MIC values ranging from 4 to 64 μg ml−1.