The geomicrobiology of limestone, sulfuric acid speleogenetic, and volcanic caves: basic concepts and future perspectives.

Caves are ubiquitous subterranean voids, accounting for a still largely unexplored surface of the Earth underground. Due to the absence of sunlight and physical segregation, caves are naturally colonized by microorganisms that have developed distinctive capabilities to thrive under extreme conditions of darkness and oligotrophy. Here, the microbiomes colonizing three frequently studied cave types, i.e., limestone, sulfuric acid speleogenetic (SAS), and lava tubes among volcanic caves, have comparatively been reviewed. Geological configurations, nutrient availability, and energy flows in caves are key ecological drivers shaping cave microbiomes through photic, twilight, transient, and deep cave zones. Chemoheterotrophic microbial communities, whose sustenance depends on nutrients supplied from outside, are prevalent in limestone and volcanic caves, while elevated inorganic chemical energy is available in SAS caves, enabling primary production through chemolithoautotrophy. The 16S rRNA-based metataxonomic profiles of cave microbiomes were retrieved from previous studies employing the Illumina platform for sequencing the prokaryotic V3-V4 hypervariable region to compare the microbial community structures from different cave systems and environmental samples. Limestone caves and lava tubes are colonized by largely overlapping bacterial phyla, with the prevalence of Pseudomonadota and Actinomycetota, whereas the co-dominance of Pseudomonadota and Campylobacterota members characterizes SAS caves. Most of the metataxonomic profiling data have so far been collected from the twilight and transient zones, while deep cave zones remain elusive, deserving further exploration. Integrative approaches for future geomicrobiology studies are suggested to gain comprehensive insights into the different cave types and zones. This review also poses novel research questions for unveiling the metabolic and genomic capabilities of cave microorganisms, paving the way for their potential biotechnological applications.

Production and characterization of lipopeptide biosurfactant from a new strain of Pseudomonas antarctica 28E using crude glycerol as a carbon source.

Pseudomonas is a cosmopolitan genus of bacteria found in soil, water, organic matter, plants and animals and known for the production of glycolipid and lipopeptide biosurfactants. In this study bacteria (laboratory collection number 28E) isolated from soil collected in Spitsbergen were used for biosurfactant production. 16S rRNA sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) revealed that this isolate belongs to the species Pseudomonas antarctica. In the present study, crude glycerol, a raw material obtained from several industrial processes, was evaluated as a potential low-cost carbon source to reduce the costs of lipopeptide production. Among several tested glycerols, a waste product of stearin production, rich in nitrogen, iron and calcium, ensured optimal conditions for bacterial growth. Biosurfactant production was evidenced by a reduction of surface tension (ST) and an increase in the emulsification index (E24%). According to Fourier-transform infrared spectroscopy (FTIR) and electrospray ionization mass spectrometry (ESI-MS), the biosurfactant was identified as viscosin. The critical micelle concentration (CMC) of lipopeptide was determined to be 20 mg L-1. Interestingly, viscosin production has been reported previously for Pseudomonas viscosa, Pseudomonas fluorescens and Pseudomonas libanensis. To the best of our knowledge, this is the first report on viscosin production by a P. antarctica 28E. The results indicated the potential of crude glycerol as a low-cost substrate to produce a lipopeptide biosurfactant with promising tensioactive and emulsifying properties.

Remediation of soil polluted with petroleum hydrocarbons and its reuse for agriculture: Recent progress, challenges, and perspectives.

Petroleum hydrocarbons (PHs) are used as raw materials in many industries and primary energy sources. However, excessive PHs act as soil pollutants, posing serious threats to living organisms. Various ex-situ or in-situ chemical and biological methods are applied to restore polluted soil. However, most of the chemical treatment methods are expensive, environmentally unfriendly, and sometimes inefficient. That attracts scientists and researchers to develop and select new strategists to remediate polluted soil through risk-based analysis and eco-friendly manner. This review discusses the sources of PHs, properties, distribution, transport, and fate in the environment, internal and external factors affecting the soil remediation and restoration process, and its effective re-utilization for agriculture. Bioremediation is an eco-friendly method for degrading PHs, specifically by using microorganisms. Next-generation sequencing (NGS) technologies are being used to monitor contaminated sites. Currently, these new technologies have caused a paradigm shift by giving new insights into the microbially mediated biodegradation processes by targeting rRNA are discussed concisely. The recent development of risk-based management for soil contamination and its challenges and future perspectives are also discussed. Furthermore, nanotechnology seems very promising for effective soil remediation, but its success depends on its cost-effectiveness. This review paper suggests using bio-electrochemical systems that utilize electro-chemically active microorganisms to remediate and restore polluted soil with PHs that would be eco-friendlier and help tailor-made effective and sustainable remediation technologies.

Isolation and characterization of a novel rhamnolipid producer Pseudomonas sp. LGMS7 from a highly contaminated site in Ain El Arbaa region of Ain Temouchent, Algeria.

This study aims to isolate and characterize a novel rhamnolipid producer within the recent bioremediation approaches for treating hydrocarbon-contaminated soils in Algeria. In this context, from a hydrocarbon-contaminated soil, a newly bacterium designated LGMS7 was screened and identified, belonged to the Pseudomonas genus, and was closely related to Pseudomonas mucidolens, with a 16S rRNA sequence similarity of 99.05%. This strain was found to use different hydrocarbons and oils as a sole carbon and energy source for growth. It showed a stable emulsification index E24 (%) of 66.66% ± 3.46 when growing in mineral salts medium (MSM) supplemented with 2% (v/v) glycerol after incubation for 6 days at 30 °C. Interestingly, it was also able to reduce the surface tension of the cell-free supernatant to around 30 ± 0.65 mN m-1 with a critical micelle concentration (CMC) of 800 mg l-1. It was found to be able to produce around 1260 ± 0.57 mg l-1 as the yield of rhamnolipid production. Its biosurfactant has demonstrated excellent stability against pH (pH 2.0-12.0), salinity (0-150 g l-1), and temperature (-20 to 121 °C). Based on various chromatographic and spectroscopic techniques (i.e., TLC, FTIR, 1H-NMR), it was found to belong to the glycolipid class (i.e., rhamnolipids). Taken altogether, the strain LGMS7 and its biosurfactant display interesting biotechnological capabilities for the bioremediation of hydrocarbon-contaminated sites. To the best of our knowledge, this is the first study that described the production of biosurfactants by Pseudomonas mucidolens species. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02751-6.

Burkholderia thailandensis E264 as a promising safe rhamnolipids' producer towards a sustainable valorization of grape marcs and olive mill pomace.

Within the circular economy framework, our study aims to assess the rhamnolipid production from winery and olive oil residues as low-cost carbon sources by nonpathogenic strains. After evaluating various agricultural residues from those two sectors, Burkholderia thailandensis E264 was found to use the raw soluble fraction of nonfermented (white) grape marcs (NF), as the sole carbon and energy source, and simultaneously, reducing the surface tension to around 35 mN/m. Interestingly, this strain showed a rhamnolipid production up to 1070 mg/L (13.37 mg/g of NF), with a higher purity, on those grape marcs, predominately Rha-Rha C14-C14, in MSM medium. On olive oil residues, the rhamnolipid yield of using olive mill pomace (OMP) at 2% (w/v) was around 300 mg/L (15 mg/g of OMP) with a similar CMC of 500 mg/L. To the best of our knowledge, our study indicated for the first time that a nonpathogenic bacterium is able to produce long-chain rhamnolipids in MSM medium supplemented with winery residues, as sole carbon and energy source. KEY POINTS: • Winery and olive oil residues are used for producing long-chain rhamnolipids (RLs). • Both higher RL yields and purity were obtained on nonfermented grape marcs as substrates. • Long-chain RLs revealed stabilities over a wide range of pH, temperatures, and salinities.

Isolation and characterization of a newly naphthalene-degrading Halomonas pacifica, strain Cnaph3: biodegradation and biosurfactant production studies.

A newly marine Halomonas pacifica strain Cnaph3 was isolated, as a naphthalene degrader and biosurfactant producer, from contaminated seawater collected in Ataya's fishing harbor, located in Kerkennah Islands, Tunisia. Chromatography flame ionization detector analysis revealed that 98.8% of naphthalene (200 mg/L) was degraded after 7 days of incubation, at 30 g/L NaCl and 37 °C. Strain Cnaph3 showed also a noticeable capacity to grow on a wide range of aliphatic, aromatic, and complex hydrocarbons. Interestingly, strain Cnaph3 showed a significant potential to produce biosurfactants in the presence of all tested substrates, particularly on glycerol (1%, v/v). Electrospray ionization analysis of the biosurfactant, designated Bios-Cnaph3, suggested a lipopeptide composition. The critical micelle concentration of Bios-Cnaph3 was about 500 mg/L. At this concentration, the surface tension of the water was reduced to 27.6 mN/m. Furthermore, Bios-Cnaph3 displayed interesting stabilities over a wide range of temperatures (4-105 °C), salinities (0-100 g/L NaCl), and pH (2.2-12.5). In addition, it showed promising capacities to remove used motor oil from contaminated soils. The biodegradation and biosurfactant-production potential of the Halomonas sp. strain Cnaph3 would present this strain as a favorite agent for bioremediation of hydrocarbon-contaminated sites under saline conditions.

Production, characterization and biotechnological potential of lipopeptide biosurfactants from a novel marine Bacillus stratosphericus strain FLU5.

This work aimed at studying the potential of a new hydrocarbonoclastic marine bacterium, Bacillus stratosphericus FLU5, to produce an efficient surface-active agent BS-FLU5. Biosurfactant production was examined on different carbon sources; using the surface tension measurement and the oil displacement test. Strain FLU5 showed its capacity to produce biosurfactants from all tested substrates, in particular the residual frying oil, which is a cheap renewable carbon source alternative, thus minimizing the high cost of producing those surfactants. MALDI-TOF MS/MS analysis confirmed the presence of lipopeptides, which are identified as members of surfactin and pumilacidin series. The critical micelle concentration (CMC) of the purified lipopeptides produced by strain FLU5 was 50 mg/l. At this concentration, the surface tension of the water was reduced from 72 to 28 mN/m. Furthermore, the crude lipopeptides showed an interesting stability against a broad range of pH, temperature and salinity. In addition, the application of BS-FLU5 in oil recovery from hydrocarbons-contaminated soil (used motor oil) showed that it was more effective on the hydrocarbon-remobilization than some tested synthetic surfactants. Interestingly, the biosurfactant BS-FLU5 showed a negligible cytotoxic effect against the mammalian cells HEK293. These results highlight the applicability of the lipopeptides BS-FLU5 in different fields, especially in environmental remediation processes.

A non-toxic microbial surfactant from Marinobacter hydrocarbonoclasticus SdK644 for crude oil solubilization enhancement.

This study aims to investigate the ability of a biosurfactant produced by Marinobacter hydrocarbonoclasticus strain SdK644 isolated from hydrocarbon contaminated sediment to enhance the solubilization rate of crude oil contaminated seawater. Phylogenetic analysis shows that strain SdK644 was very closely related to M. hydrocarbonoclasticus with 16S rRNA gene sequence similarity of 97.44%. Using waste frying oil as inducer carbon source, the producing biosurfactant by strain SdK644 was applied to improve crude oil solubilization in seawater. The preliminary characterization of the produced biosurfactant by FT-IR analysis indicates its possible classification in a glycolipids group. Results from crude oil solubilization assay showed that SdK644 strain biosurfactant was 2-fold greater than Tween 80 surfactant in crude oil solubilization and 12-fold higher than seawater control, as shown by GC-MS analysis of aliphatic compounds. Furthermore, this bioactive compound was shown to be nontoxic against Artemia larvae in short-term acute toxicity bioassay. Generally, the results showed the possible use of M. hydrocarbonoclasticus strain SdK644 biosurfactant in bioremediation processes of the marine environments.

Optimized production and characterization of a detergent-stable protease from Lysinibacillus fusiformis C250R.

In this study, we aimed to optimize the cultural and nutritional conditions for protease production by Lysinibacillus fusiformis strain C250R in submerged fermentation process using statistical methodology. The most significant factors (gruel, wheat bran, yeast extract, and FeSO4) were identified by Plackett-Burman design. Response surface methodology (RSM) was used to determine the optimum levels of the screened factors and their interaction. Under the optimized conditions, protease yield 3100U/mL was 4.5 folds higher than those obtained by the use of the initial conditions (680U/mL). Additionally, a new extracellular 51kDa-protease, designated SAPLF, was purified and biochemically characterized from strain C250R. It shows optimum activity at 70°C and pH 10. Its half-life times at 70 and 80°C were 10 and 6-h, respectively. Irreversible inhibition of enzyme activity of SAPLF with serine protease inhibitors demonstrated that it belongs to the serine protease family. Interestingly, its catalytic efficiency was higher than that of SPVP from Aeribacillus pallidus strain VP3 and Alcalase Ultra 2.5L from Bacillus licheniformis. This study demonstrated that SAPLF has a high detergent compatibility and an excellent stain removal compared to Alcalase Ultra 2.5L; which offers an interesting potential for its application in the laundry detergent industry.

Rhamnolipids from Pseudomonas aeruginosa strain W10; as antibiofilm/antibiofouling products for metal protection.

Industrial biofouling-problems associated with the accumulation of microorganisms from flowing water and fluids on processing surfaces can cause severe problems. A Pseudomonas aeruginosa strain W10 was isolated from industrial setting and found to produce predominantly di-rhamnolipids (Rha-Rha-C10-C10) with a yield of around 10 g L-1 and a critical micelle concentration (CMC) of 80 mg L-1 . P. aeruginosa W10 rhamnolipids were able to disrupt up to 99% of 48 h pre-formed biofilms of the Gram-positive organisms Bacillus licheniformis CAN55, Staphylococcus capitis SH6, and a mixed culture (strains CAN55, SH6, and W10), under static conditions, at concentrations of 0.1, 0.5, and 1 mg ml-1 on a stainless steel surface commonly used in industrial process pipelines. CFU measurements and LIVE/DEAD BacLight staining confirmed these observations. Furthermore, a purified di-rhamnolipid fraction was found to be responsible for the microbial inhibition of B. licheniformis strain CAN55. This study provides evidence that rhamnolipids may have valuable applications in preventing biofilms and biofouling in industrial plants and, in a wider context, may also apply to metal medical devices.

Rhamnolipids from non-pathogenic Burkholderia thailandensis E264: Physicochemical characterization, antimicrobial and antibiofilm efficacy against oral hygiene related pathogens.

Biosurfactants are naturally occurring surface active compounds that have mainly been exploited for environmental applications and consumer products, with their biomedical efficacy an emerging area of research. Rhamnolipids area major group of biosurfactants that have been reported for their antimicrobial and antibiofilm efficacy. One of the main limiting factors for scaled up production and downstream applications of rhamnolipids is the fact that they are predominantly produced from the opportunistic pathogen Pseudomonas aeruginosa. In this article, we have reported the production and characterisation of long chain rhamnolipids from non-pathogenic Burkholderia thailandensis E264 (ATCC 700388). We have also investigated the antibacterial and antibiofilm properties of these rhamnolipids against some oral pathogens (Streptococcus oralis, Actinomyces naeslundii, Neisseria mucosa and Streptococcus sanguinis), important for oral health and hygiene. Treating these bacteria with different concentrations of long chain rhamnolipids resulted in a reduction of 3-4 log of bacterial viability, placing these rhamnolipids close to being classified as biocidal. Investigating long chain rhamnolipid efficacy as antibiofilm agents for prospective oral-related applications revealed good potency against oral-bacteria biofilms in a co-incubation experiments, in a pre-coated surface format, in disrupting immature biofilms and has shown excellent combination effect with Lauryl Sodium Sulphate which resulted in a drastic decrease in its minimal inhibitory concentration against different bacteria. Investigating the rhamnolipid permeabilization effect along with their ability to induce the formation of reactive oxygen species has shed light on the mechanism through which inhibition/killing of bacteria may occur.

Characterization of a novel protease from Aeribacillus pallidus strain VP3 with potential biotechnological interest.

The present study investigates the purification and physico-chemical characterization of an extracellular protease from the Aeribacillus pallidus strain VP3 previously isolated from a geothermal oil-field (Sfax, Tunisia). The maximum protease activity recorded after 22h of incubation at 45°C was 3000U/ml. Pure enzyme, designated as SPVP, was obtained after ammonium sulfate fractionation (40-60%)-dialysis followed by heat-treatment (70°C for 30min) and UNO Q-6 FPLC anion-exchange chromatography. The purified enzyme is a monomer of molecular mass about 29kDa. The sequence of the 25 NH2-terminal residues of SPVP showed a high homology with those of Bacillus proteases. The almost complete inhibition by PMSF and DIFP confirmed that SPVP is a member of serine protease family. Its optima of pH and temperature were pH 10 and 60°C, respectively. Its half-life times at 70 and 80°C were 8 and 4h, respectively. Its catalytic efficiency was higher than those of SAPCG, Alcalase Ultra 2.5L, and Thermolysin type X. SPVP exhibited excellent stability to detergents and wash performance analysis revealed that it could remove blood-stains effectively and high resistance against organic solvents. These properties make SPVP a potential candidate for applications in detergent formulations and non-aqueous peptide biocatalysis.

Preliminary characterization of biosurfactant produced by a PAH-degrading Paenibacillus sp. under thermophilic conditions.

The capacities of a biosurfactant producing and polycyclic aromatic hydrocarbon (PAH) utilizing bacterium, namely, strain 1C, isolated from an Algerian contaminated soil, were investigated. Strain 1C belonged to the Paenibacillus genus and was closely related to the specie Paenibacillus popilliae, with 16S rRNA gene sequence similarity of 98.4 %. It was able to produce biosurfactant using olive oil as substrate. The biosurfactant production was shown by surface tension (32.6 mN/m) after 24 h of incubation at 45 °C and 150 rpm. The biosurfactant(s) retained its properties during exposure to elevated temperatures (70 °C), relatively high salinity (20 % NaCl), and a wide range of pH values (2-10). The infrared spectroscopy (FTIR) revealed that its chemical structure belonged to lipopeptide class. The critical micelle concentration (CMC) of this biosurfactant was about 0.5 g/l with 29.4 mN/m. In addition, the surface active compound(s) produced by strain 1C enhanced PAH solubility and showed a significant antimicrobial activity against pathogens. In addition to its biosurfactant production, strain 1C was shown to be able to utilize PAHs as the sole carbon and energy sources. Strain 1C as hydrocarbonoclastic bacteria and its interesting surface active agent may be used for cleaning the environments polluted with polyaromatic hydrocarbons.

Biodegradation of fluoranthene by a newly isolated strain of Bacillus stratosphericus from Mediterranean seawater of the Sfax fishing harbour, Tunisia.

A physico-chemical characterization of seawater taken from the fishing harbour of Sfax, Tunisia, revealed a contamination by organic and inorganic micropollutants. An aerobic marine halotolerant Bacillus stratosphericus strain FLU5 was isolated after enrichment on fluoranthene, a persistent and toxic polycyclic aromatic hydrocarbon (PAH). GC-MS analyses showed that strain FLU5 was capable of degrading almost 45 % of fluoranthene (100 mg l(-1)), without yeast extract added, after 30 days of incubation at 30 g l(-1) NaCl and 37 °C. In addition, the isolate FLU5 showed a remarkable capacity to grow on a wide range of aliphatic, aromatic and complex hydrocarbons. This strain could also synthesize a biosurfactant which was capable of reducing the surface tension of the cell-free medium, during the growth on fluoranthene. The biodegradative abilities of PAHs are promising and can be used to perform the bioremediation strategies of seawaters and marine sediments contaminated by hydrocarbons.

Biodegradation of malodorous thiols by a Brevibacillus sp. strain isolated from a Tunisian phosphate factory.

Hydrogen sulfide (H2S) and thiols (RSH) generated by the phosphate industry cause harmful effects on human health and quality of life. The present study aims to investigate and evaluate a bacterial strain CAT37 isolated from gas-washing wastewaters in terms of its properties and ability to degrade malodorous thiols. Gas-washing wastewater samples were submitted to physicochemical analyses and used for the isolation of thiol-degrading bacteria. The results from gas chromatography-mass spectrometry (GC-MS) analysis revealed that the isolated strain CAT37 was able to oxidize ∼99% of each thiol, decanethiol and dodecanethiol used as sole carbon and energy sources after 30 days of incubation at 37°C. The strain CAT37 displayed a biodegradative potential on several thiols known by their toxicity and odors. The results from phylogenetic and phenotypic analysis revealed that the CAT37 isolate belonged to the genus Brevibacillus, showing the highest sequence similarity to Brevibacillus agri. Overall, the results indicated that the strain CAT37 exhibited a number of attractive biodegradation abilities against thiols and could be considered a promising candidate for industrial application in future thiol biodeodorization strategies.