Pre-incubation conditions determine the fermentation pattern and microbial community structure in fermenters at mild hydrostatic pressure.

Fermentation at elevated hydrostatic pressure is a novel strategy targeting product selectivity. However, the role of inoculum history and cross-resistance, that is, acquired tolerance from incubation under distinctive environmental stress, remains unclear in high-pressure operation. In our here presented work, we studied fermentation and microbial community responses of halotolerant marine sediment inoculum (MSI) and anaerobic digester inoculum (ADI), pre-incubated in serum bottles at different temperatures and subsequently exposed to mild hydrostatic pressure (MHP; < 10 MPa) in stainless steel reactors. Results showed that MHP effects on microbial growth, activity, and community structure were strongly temperature-dependent. At moderate temperature (20°C), biomass yield and fermentation were not limited by MHP; suggesting a cross-resistance effect from incubation temperature and halotolerance. Low temperatures (10°C) and MHP imposed kinetic and bioenergetic limitations, constraining growth and product formation. Fermentation remained favorable in MSI at 28°C and ADI at 37°C, despite reduced biomass yield resulting from maintenance and decay proportionally increasing with temperature. Microbial community structure was modified by temperature during the enrichment, and slight differences observed after MHP-exposure did not compromise functionality. Results showed that the relation incubation temperature-halotolerance proved to be a modifier of microbial responses to MHP and could be potentially exploited in fermentations to modulate product/biomass ratio.

Antimicrobial potential and taxonomic investigation of piezotolerant Streptomyces sp. NIOT-Ch-40 isolated from deep-sea sediment.

Microbial-derived natural products from extreme niches such as deepsea are known to possess structural and functional novelty. With this background, the present study was designed to investigate the bioprospecting potential and systematics of a deep-sea derived piezotolerant bacterial strain NIOT-Ch-40, showing affiliation to the genus Streptomyces based on 16S RNA gene similarity. Preliminary screening for the presence of biosynthetic genes like polyketide synthase I, polyketide synthase II, non ribosomal peptide synthase, 3-amino-5-hydroxybenzoic acid synthase and spiroindimicin followed by antibacterial activity testing confirmed the presence of potent bioactivity. The secondary metabolites produced during fermentation in Streptomyces broth at 28 °C for 7 days were extracted with ethyl acetate. The extract exhibited a specific inhibitory activity against Gram-positive bacteria and was significantly effective (p < 0.0001) against methicillin-resistant Staphylococcus aureus (MRSA). The minimum inhibitory concentration and minimum bactericidal concentration against MRSA was 1.5 µg/mL, which was statistically significant in comparison with erythromycin. A multifaceted analysis of the Streptomyces spp. was carried out to delineate the strain NIOT-Ch-40 at a higher resolution which includes morphological, biochemical and molecular studies. Piezotolerance studies and comparison of fatty acid profiles at high pressures revealed that it could be considered as one of the taxonomic markers, especially for the strains isolated from the deep sea environments. In conclusion, the observation of comparative studies with reference strains indicated towards the strain NIOT-Ch-40 as an indigenous marine piezotolerant Streptomyces sp. with a higher probability of obtaining novel bioactive metabolites.

Insight into the adaptation mechanisms of high hydrostatic pressure in physiology and metabolism of hadal fungi from the deepest ocean sediment.

High hydrostatic pressure (HHP) influences the life processes of organisms living at depth in the oceans. While filamentous fungi are one of the essential members of deep-sea microorganisms, few works have explored their piezotolerance to HHP. Here, we obtained three homogeneous Aspergillus sydowii from terrestrial, shallow, and hadal areas, respectively, to compare their pressure resistance. A set of all-around evaluation methods including determination of growth rate, metabolic activity, and microscopic staining observation was established and indicated that A. sydowii DM1 from the hadal area displayed significant piezotolerance. Global analysis of transcriptome data under elevated HHP revealed that A. sydowii DM1 proactively modulated cell membrane permeability, hyphae morphology, and septal quantities for seeking a better livelihood under mild pressure. Besides, differentially expressed genes were mainly enriched in the biosynthesis of amino acids, carbohydrate metabolism, cell process, etc., implying how the filamentous fungi respond to elevated pressure at the molecular level. We speculated that A. sydowii DM1 could acclimatize itself to HHP by adopting several strategies, including environmental response pathway HOG-MAPK, stress proteins, and cellular metabolisms.IMPORTANCEFungi play an ecological and biological function in marine environments, while the physiology of filamentous fungi under high hydrostatic pressure (HHP) is an unknown territory due to current technologies. As filamentous fungi are found in various niches, Aspergillus sp. from deep-sea inspire us to the physiological trait of eukaryotes under HHP, which can be considered as a prospective research model. Here, the evaluation methods we constructed would be universal for most filamentous fungi to assess their pressure resistance, and we found that Aspergillus sydowii DM1 from the hadal area owned better piezotolerance and the active metabolisms under HHP indicated the existence of undiscovered metabolic strategies for hadal fungi. Since pressure-related research of marine fungi has been unexpectedly neglected, our study provided an enlightening strategy for them under HHP; we believed that understanding their adaptation and ecological function in original niches will be accelerated in the perceivable future.

Importance of Intracellular Energy Status on High-Hydrostatic-Pressure Inactivation of sake Yeast Saccharomyces cerevisiae.

The HHP inactivation behaviors of Niigata sake yeast Saccharomyces cerevisiae strain S9arg and its aerobic respiratory-deficient mutant strains were investigated after cultivating them in a YPD media containing 2% to 15% glucose, as well as in moromi mash, in a laboratory-scale sake brewing process. The piezotolerance of strain S9arg, shown after cultivation in a YPD medium containing 2% glucose, decreased to become piezosensitive with increasing glucose concentrations in YPD media. In contrast, the piezosensitivity of a mutant strain UV1, shown after cultivation in the YPD medium containing 2% glucose, decreased to become piezotolerant with increasing glucose concentrations in the YPD medium. The intracellular ATP concentrations were analyzed for an S. cerevisiae strain with intact aerobic respiratory ability, as well as for strain UV1. The higher concentration of ATP after cultivation suggested a higher energy status and may be closely related to higher piezotolerance for the yeast strains. The decreased piezotolerance of strain S9arg observed after a laboratory-scale sake brewing test may be due to a lower energy status resulting from a high glucose concentration in moromi mash during the early period of brewing, as well as a lower aeration efficiency during the brewing process, compared with cultivation in a YPD medium containing 2% glucose.

High hydrostatic pressure harnesses the biosynthesis of secondary metabolites via the regulation of polyketide synthesis genes of hadal sediment-derived fungi.

Deep-sea fungi have evolved extreme environmental adaptation and possess huge biosynthetic potential of bioactive compounds. However, not much is known about the biosynthesis and regulation of secondary metabolites of deep-sea fungi under extreme environments. Here, we presented the isolation of 15 individual fungal strains from the sediments of the Mariana Trench, which were identified by internal transcribed spacer (ITS) sequence analysis as belonging to 8 different fungal species. High hydrostatic pressure (HHP) assays were performed to identify the piezo-tolerance of the hadal fungi. Among these fungi, Aspergillus sydowii SYX6 was selected as the representative due to the excellent tolerance of HHP and biosynthetic potential of antimicrobial compounds. Vegetative growth and sporulation of A. sydowii SYX6 were affected by HHP. Natural product analysis with different pressure conditions was also performed. Based on bioactivity-guided fractionation, diorcinol was purified and characterized as the bioactive compound, showing significant antimicrobial and antitumor activity. The core functional gene associated with the biosynthetic gene cluster (BGC) of diorcinol was identified in A. sydowii SYX6, named as AspksD. The expression of AspksD was apparently regulated by the HHP treatment, correlated with the regulation of diorcinol production. Based on the effect of the HHP tested here, high pressure affected the fungal development and metabolite production, as well as the expression level of biosynthetic genes which revealed the adaptive relationship between the metabolic pathway and the high-pressure environment at the molecular level.

Microbial enrichment, functional characterization and isolation from a cold seep yield piezotolerant obligate hydrocarbon degraders.

Deep-sea environments can become contaminated with petroleum hydrocarbons. The effects of hydrostatic pressure (HP) in the deep sea on microbial oil degradation are poorly understood. Here, we performed long-term enrichments (100 days) from a natural cold seep while providing optimal conditions to sustain high hydrocarbon degradation rates. Through enrichments performed at increased HP and ambient pressure (AP) and by using control enrichments with marine broth, we demonstrated that both pressure and carbon source can have a big impact on the community structure. In contrast to previous studies, hydrocarbonoclastic operational taxonomic units (OTUs) remained dominant at both AP and increased HP, suggesting piezotolerance of these OTUs over the tested pressure range. Twenty-three isolates were obtained after isolation and dereplication. After recultivation at increased HP, an Alcanivorax sp. showed promising piezotolerance in axenic culture. Furthermore, preliminary co-cultivation tests indicated synergistic growth between some isolates, which shows promise for future synthetic community construction. Overall, more insights into the effect of increased HP on oil-degrading communities were obtained as well as several interesting isolates, e.g. a piezotolerant hydrocarbonoclastic bacterium for future deep-sea bioaugmentation investigation.

Pressure-Regulated Gene Expression and Enzymatic Activity of the Two Periplasmic Nitrate Reductases in the Deep-Sea Bacterium Shewanella piezotolerans WP3.

Shewanella species are widely distributed in marine environments, from the shallow coasts to the deepest sea bottom. Most Shewanella species possess two isoforms of periplasmic nitrate reductases (NAP-α and NAP-ß) and are able to generate energy through nitrate reduction. However, the contributions of the two NAP systems to bacterial deep-sea adaptation remain unclear. In this study, we found that the deep-sea denitrifier Shewanella piezotolerans WP3 was capable of performing nitrate respiration under high hydrostatic pressure (HHP) conditions. In the wild-type strain, NAP-ß played a dominant role and was induced by both the substrate and an elevated pressure, whereas NAP-α was constitutively expressed at a relatively lower level. Genetic studies showed that each NAP system alone was sufficient to fully sustain nitrate-dependent growth and that both NAP systems exhibited substrate and pressure inducible expression patterns when the other set was absent. Biochemical assays further demonstrated that NAP-α had a higher tolerance to elevated pressure. Collectively, we report for the first time the distinct properties and contributions of the two NAP systems to nitrate reduction under different pressure conditions. The results will shed light on the mechanisms of bacterial HHP adaptation and nitrogen cycling in the deep-sea environment.

Comparative analysis on inactivation kinetics of between piezotolerant and piezosensitive mutant strains of Saccharomyces cerevisiae under combinations of high hydrostatic pressure and temperature.

We previously obtained a pressure-tolerant (piezotolerant) and a pressure sensitive (piezosensitive) mutant strain, under ambient temperature, from Saccharomyces cerevisiae strain KA31a. The inactivation kinetics of these mutants were analyzed at 150 to 250MPa with 4 to 40°C. By a multiple regression analysis, the pressure and temperature dependency of the inactivation rate constants k values of both mutants, as well as the parent strain KA31a, were well approximated with high correlation coefficients (0.92 to 0.95). For both mutants, as well as strain KA31a, the lowest k value was shown at a low pressure levels with around ambient temperature. The k value approximately increased with increase in pressure level, and with increase and decrease in temperature. The piezosensitive mutant strain a924E1 showed piezosensitivity at all pressure and temperature levels, compared with the parent strain KA31a. In contrast, the piezotolerant mutant strain a2568D8 showed piezotolerance at 4 to 20°C, but did not show significant piezotolerance at 40°C. These results of the variable influence of temperature on pressure inactivation of these strains would be important for better understanding of piezosensitive and piezotolerant mechanisms, as well as the pressure inactivation mechanism of S. cerevisiae.

Effects of hydrostatic pressure on yeasts isolated from deep-sea hydrothermal vents.

Hydrostatic pressure plays a significant role in the distribution of life in the biosphere. Knowledge of deep-sea piezotolerant and (hyper)piezophilic bacteria and archaea diversity has been well documented, along with their specific adaptations to cope with high hydrostatic pressure (HHP). Recent investigations of deep-sea microbial community compositions have shown unexpected micro-eukaryotic communities, mainly dominated by fungi. Molecular methods such as next-generation sequencing have been used for SSU rRNA gene sequencing to reveal fungal taxa. Currently, a difficult but fascinating challenge for marine mycologists is to create deep-sea marine fungus culture collections and assess their ability to cope with pressure. Indeed, although there is no universal genetic marker for piezoresistance, physiological analyses provide concrete relevant data for estimating their adaptations and understanding the role of fungal communities in the abyss. The present study investigated morphological and physiological responses of fungi to HHP using a collection of deep-sea yeasts as a model. The aim was to determine whether deep-sea yeasts were able to tolerate different HHP and if they were metabolically active. Here we report an unexpected taxonomic-based dichotomic response to pressure with piezosensitve ascomycetes and piezotolerant basidiomycetes, and distinct morphological switches triggered by pressure for certain strains.