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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 have several applications in nutraceuticals, pharmaceuticals, cosmetics, biofuel production, and bioremediation, among other fields. Isolation and purification are extremely important for obtaining axenic cultures of microalgae from different environments and crucial for their biotechnological applications, but it is not an easy task. In view of the above, it is fundamental to know the classical and advanced techniques and examples of how scientists from around the globe have applied such methods to isolate several genera and the impact of each step on successful algal purification. This review provides a brief and simple explanation of the methodology for sampling, growth, obtention of unialgal, and posterior axenic culture, which will facilitate the development of novel microalgae-related discoveries and applications for new researchers.

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.

Ectomycorrhizal fungi contribute to the nutrition of many woody plants, including those in the Pinaceae family. Loblolly pine (Pinus taeda L.), a native species of the Southeastern USA, can be colonized by multiple species of ectomycorrhizal fungi. The role of these symbionts in P. taeda potassium (K+) nutrition has not been previously investigated. Here, we assessed the contribution of four ectomycorrhizal fungi, Hebeloma cylindrosporum, Paxillus ammoniavirescens, Laccaria bicolor, and Suillus cothurnatus, in P. taeda K+ acquisition under different external K+ availabilities. Using a custom-made two-compartment system, P. taeda seedlings were inoculated with one of the four fungi, or kept non-colonized, and grown under K+-limited or -sufficient conditions for 8 weeks. Only the fungi had access to separate compartments in which rubidium, an analog tracer for K+, was supplied before harvest. Resulting effects of the fungi were recorded, including root colonization, biomass, and nutrient concentrations. We also analyzed the fungal performance in axenic conditions under varying supply of K+ and sodium. Our study revealed that these four ectomycorrhizal fungi are differentially affected by external K+ and sodium variations, that they are not able to provide similar benefits to the host P. taeda in our growing conditions, and that rubidium may be used with some limitations to estimate K+ transport from ectomycorrhizal fungi to colonized plants.

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.

Objective: Free-living amoeba (FLA) are protozoa living in soil and in natural and man-made water systems. They attract much attention owing to the illnesses associated with them and to their relationships with bacteria. In this study, the effect of cell-free supernatant (CFS) obtained from FLA against Staphylococcus was investigated. Materials and Methods: Environmental FLA strains (A1, A2, A3) were obtained from lake water and swimming pools in Istanbul. Acanthamoeba castellanii ATCC 50373 was used as the standard strain. Clinical Staphylococcus strains (S1, S2, S3) were obtained from a culture collection at Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology. As standard strains, MRSA ATCC 43300, S. epidermidis ATCC 12228, S. aureus ATCC 29213 were used. FLA-CFS were obtained by centrifuging and filtering of axenic cultures. Colony counting technique was used to investigate the inhibition activities of FLA-CFS against Staphylococcus bacteria. Results: Against MRSA ATCC 43300 strain, CFSs of A. castellanii ATCCC 50373 and A1 showed an inhibition efficiency of 78.36% and 73.47%, respectively. Against S1 strain, CFSs of A. castellanii ATCCC 50373 and A2 showed an inhibition of 65.64% and 15.14%, respectively. Against S. aureus ATCC 29213, only A1-CFS showed inhibitory effect (44%). It was found that A. castellanii ATCC 50373 and A2-CFSs inhibited the S2 strain 26.20% and 9.24% respectively. Against S3 strain, A2-CFS was inhibitory at 33.33%. No FLA-CFS could be inhibitory against S. epidermidis ATCC 12228. Conclusion: It is necessary to devise new studies in which sample numbers are increased when using FLA strains in the inhibition of antibiotic-resistant bacteria.

The mucosa-associated microbiota is widely recognized as a potential trigger for Crohn’s disease pathophysiology but remains largely uncharacterised beyond its taxonomic composition. Unlike stool microbiota, the functional characterisation of these communities using current DNA/RNA sequencing approaches remains constrained by the relatively small microbial density on tissue, and the overwhelming amount of human DNA recovered during sample preparation. Here, we have used a novel ex vivo approach that combines microbe culture from anaerobically preserved tissue with metagenome sequencing (MC-MGS) to reveal patient-specific and strain-level differences among these communities in post-operative Crohn’s disease patients. The 16 S rRNA gene amplicon profiles showed these cultures provide a representative and holistic representation of the mucosa-associated microbiota, and MC-MGS produced both high quality metagenome-assembled genomes of recovered novel bacterial lineages. The MC-MGS approach also produced a strain-level resolution of key Enterobacteriacea and their associated virulence factors and revealed that urease activity underpins a key and diverse metabolic guild in these communities, which was confirmed by culture-based studies with axenic cultures. Collectively, these findings using MC-MGS show that the Crohn’s disease mucosa-associated microbiota possesses taxonomic and functional attributes that are highly individualistic, borne at least in part by novel bacterial lineages not readily isolated or characterised from stool samples using current sequencing approaches.

Acanthamoeba spp. is one of the free-living amoebas in the environment that many studies focused on. This parasite is of veterinary and public health importance. The present study aimed to evaluate several available media for Acanthamoeba spp. culture to find a suitable axenic liquid medium. In the present study four culture media, including TYM, RPMI-1640, and hydatid cyst fluid (HCF) as well as non-nutrient agar (NNA) as the control group, were examined to evaluate the proliferation of Acanthamoeba T4 genotype at 24, 48, and 72 h. Based on our results, at 24 and 48 h, the highest number of cysts was observed in TYM medium and the lowest in NNA medium. Moreover, at 72 h, the highest and lowest number of cysts was detected in the HCF and NNA media, respectively. In the present study, the best medium for proliferation of the parasite in the short term was TYM. Furthermore, the parasite proliferated continuously in a considerable number in HCF medium, and this medium can be used if continuative examinations are needed on the parasite.

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.

The impact of trypanosomatid parasites on honeybee health may represent a major threat to bee colonies worldwide. However, few axenic isolates have been generated to date and with no details on cell culture passages, a parameter that could influence parasite virulence. To address this question, a trypanosomatid isolation protocol was developed and a new strain was obtained, named L. passim C1. Using experimental infection of worker honeybees, we compared the virulence and mortality rates of the ATCC PRA-403 reference strain and C1 strain, the latter showing higher virulence from 10 days post-infection onward. This study highlights the impact of cell culture passages on the pathogenicity of L. passim in honeybees, providing new evidence of its negative effects on honeybee health.

Insects' intestinal microbes have profound effects on the host's physiological traits, which can impact their physiology at both the local (gut) and systemic (body) levels. Nevertheless, the molecular mechanisms underlying host-microbiota interactions, especially in non-model insects, remain elusive. Recently, tissue-specific transcriptomic analysis has been highlighted as a robust tool in studying host-microbe interactions. Plagiodera versicolora is a worldwide leaf-eating pest that primarily feeds on willows and poplar. The interplay between gut microflora and this host beetle has yet to be studied. Herein, we investigate the effects of the gut microbiota on the body mass of P. versicolora larvae, compare the nutrition status of larvae in absence and presence of gut microbiota, and profile gut bacterial loads throughout its developmental larval stages. We then perform comparative transcriptomic analysis of gut and body tissues in axenic and non-axenic larvae. Finally, we confirm the expression patterns of representative genes in nutritional metabolism and immunity. Results show that weight growth is retarded in conventional larvae, with a concomitant increase of total bacterial load by the 5th development day, and germ-free larvae have a higher glucose content than conventional-reared larvae. Both nutritional and immunological analyses indicate that gut bacteria are a burden in the beetle's larval development. These findings elucidate the impacts of gut microbiota on P. versicolora, and provide insight into tissue-specific responses to gut microflora in this pest at the genetic level, boosting our understanding of the molecular mechanisms underlying host-microbe interactions in leaf beetles and beyond.

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.

Leprosy is a chronic granulomatous infectious disease caused by the pathogen, Mycobacterium leprae, and the more recently discovered, M. lepromatosis. Described in 1873, M. leprae was among the first microorganisms to be proposed as a cause of a human infectious disease. As an obligate intracellular bacterium, it has still not thus far been reproducibly cultivated in axenic medium or cell cultures. Shepard's mouse footpad assay, therefore, was truly a breakthrough in leprosy research. The generation of immunosuppressed and genetically engineered mice, along with advances in molecular and cellular techniques, has since offered more tools for the study of the M. leprae–induced granuloma. While far from perfect, these new mouse models have provided insights into the immunoregulatory mechanisms responsible for the spectrum of this complex disease.

Nowadays, artificial construction of bacteria-algae consortia to enhance microalgal biomass is prevalent in enclosed systems, while few are built in an open culture. In this study, Achromobacter sp. and Rhizobium sp., isolated from an open pond of Chlorella sorokiniana, were the microalgal growth-promotion bacteria and selected to build the bacteria-algae consortia with axenic C. sorokiniana in open cultivation systems. To examine the performance of these two artificial bacteria-algae consortia in open culture under stable cultivation conditions, the co-cultivation experiments were conducted under constant temperature and light intensity indoors. It was found that Achromobacter sp. gradually lost the dominance of the population in the co-culture and failed to promote the growth of C. sorokiniana during open cultivation. However, the Rhizobium sp. maintained its dominant population of bacterial community in open culture and could promote the growth of C. sorokiniana, with an enhancement of 13.76%. To further evaluate the effects of Rhizobium sp. on microalgae under variations of temperature and sunlight intensity conditions, the open co-cultivation experiments were built outdoors. Results showed that the growth of C. sorokiniana could rise 13.29% only when Rhizobium sp. was added to the culture continuously, and addition of bacterial solution in log-phase of microalgae could help Rhizobium sp. dominate in the bacterial community. In this way, addition of Rhizobium sp. in the log-phase of C. sorokiniana should be an effective process to be applied to open ponds cultivation. Our findings are a step toward applying growth-promotion bacteria for C. sorokiniana for applications in open cultivation systems.

Despite the economic and ecological importance of Hydnum species, in vitro synthesis of ectomycorrhizae of this genus has not been reported due to difficulties in establishing pure cultures. We inoculated pure cultures of 12 monokaryotic and 3 dikaryotic mycelial strains of an undescribed Hydnum albidum-like species on roots of axenic Pinus densiflora seedlings to synthesize ectomycorrhizae and to evaluate their mycorrhiza-forming ability. Six months after inoculation, both monokaryotic and dikaryotic strains formed ectomycorrhizae with Hartig net hyphae at the root cortex. Monokaryotic and dikaryotic strains exhibited similar morpho-anatomical characteristics of ectomycorrhizae, with the exception for clamped septa of emanating and outer mantle hyphae in the latter. Between monokaryotic and descendant dikaryotic strains, there were no significant differences in number of mycorrhizae in pine seedlings, whereas monokaryotic strains showed a greater total number of root tips and lower colonization rates than the descendant dikaryotic strains. These results indicate that both monokaryotic and dikaryotic mycelia of the H. albidum-like species can form mycorrhizae under axenic condition, and that can be applied toward the cultivation of hedgehog mushrooms.

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.

Leishmaniasis is a tropical parasitic disease that affects up to 12 million people worldwide. Current chemotherapies have limitations such as toxicity, high cost, and parasite resistance. This work aims to select an essential oil (EssOil) isolated from the Tunisian flora as a new antileishmanial candidate. Two plants were chosen for their antileishmanial potential: Citrus limon (Citrus) and Pistacia lentiscus (Pistacia). Each of these plants was harvested from two different sites (area 1 and area 2). Extracted EssOils were characterized using GC-MS. Their antiparasitic activity against axenic and intracellular Leishmania major amastigotes and their cytotoxicity were assessed. Citrus EssOil from area 1 displayed an interesting activity against L. major intramacrophage amastigotes with IC50 value at 4.2 ± 1.3 μg/mL. Interestingly, this activity was close to that of miltefosine. Moderate activities against intracellular amastigote were observed for Pistacia EssOil from area 1 and Citrus EssOil from area 2. However, low cytotoxicity with high selectivity index was proved only for Citrus EssOil from area 1, revealing its safety for macrophages. This study also demonstrated for the first time the antileishmanial activity of EssOil extracted from Citrus limon leaves. The EssOil interesting activity could be related to the lipophilic properties of terpenes that were shown in literature to contribute to the disruption of parasite intracellular metabolic pathways.

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.

Background Nutrient stoichiometry of phytoplankton frequently changes with aquatic ambient nutrient concentrations, which is mainly influenced by anthropogenic water treatment and the ecosystem dynamics. Consequently, the stoichiometry of phytoplankton can markedly alter the metabolism and growth of zooplankton. However, the effects of nutrient-imbalanced prey on the interplay between zooplankton and their gut microbiota remain unknown. Using metatranscriptome, a 16 s rRNA amplicon-based neutral community model (NCM) and experimental validation, we investigated the interactions between Daphnia magna and its gut microbiota in a nutrient-imbalanced algal diet. Results Our results showed that in nutrient-depleted water, the nutrient-enriched zooplankton gut stimulated the accumulation of microbial polyphosphate in fecal pellets under phosphorus limitation and the microbial assimilation of ammonia under nitrogen limitation. Compared with the nutrient replete group, both N and P limitation markedly promoted the gene expression of the gut microbiome for organic matter degradation but repressed that for anaerobic metabolisms. In the nutrient limited diet, the gut microbial community exhibited a higher fit to NCM (R2 = 0.624 and 0.781, for N- and P-limitation, respectively) when compared with the Control group (R2 = 0.542), suggesting increased ambient-gut exchange process favored by compensatory feeding. Further, an additional axenic grazing experiment revealed that the growth of D. magna can still benefit from gut microbiota under a nutrient-imbalanced diet. Conclusions Together, these results demonstrated that under a nutrient-imbalanced diet, the microbes not only benefit themselves by absorbing excess nutrients inside the zooplankton gut but also help zooplankton to survive during nutrient limitation.

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.