Advances in high-throughput screening approaches for biosurfactants: current trends, bottlenecks and perspectives.

The market size of biosurfactants (BSs) has been expanding at an extremely fast pace due to their broad application scope. Therefore, the re-construction of cell factories with modified genomic and metabolic profiles for desired industrial performance has been an intriguing aspect. Typical mutagenesis approaches generate huge mutant libraries, whereas a battery of specific, robust, and cost-effective high-throughput screening (HTS) methods is requisite to screen target strains for desired phenotypes. So far, only a few specialized HTS assays have been developed for BSs that were successfully applied to obtain anticipated mutants. The most important milestones to reach, however, continue to be: specificity, sensitivity, throughput, and the potential for automation. Here, we discuss important colorimetric and fluorometric HTS approaches for possible intervention on automated HTS platforms. Moreover, we explain current bottlenecks in developing specialized HTS platforms for screening high-yielding producers and discuss possible perspectives for addressing such challenges.

Evaluation of di-rhamnolipid biosurfactants production by a novel Pseudomonas sp. S1WB: Optimization, characterization and effect on petroleum-hydrocarbon degradation.

Rhamnolipid biosurfactants are multifunctional compounds that can play an indispensable role in biotechnological, biomedical, and environmental bioremediation-related fields, and have attracted significant attention in recent years. Herein, a novel strain Pseudomonas sp. S1WB was isolated from an oil-contaminated water sample. The biosurfactants produced by this strain have capabilities to reduce surface tension (SFT) at 32.75 ± 1.63 mN/m and emulsified 50.2 ± 1.13 % in liquid media containing 1 % used engine oil (UEO) as the sole carbon source. However, the lowest SFT reduction (28.25 ± 0.21), highest emulsification index (60.15 ± 0.07), and the maximum yields (900 mg/L) were achieved under optimized conditions; where, the glucose/urea and glycerol/urea combinations were found efficient carbon and nitrogen substrates for improved biosurfactants production. Biosurfactants product was characterized using ultra-high performance liquid chromatography-mass spectrometry (UHPLC- MS) and detected various di- rhamnolipids congeners. In addition, the di-rhamnolipids produced by S1WB strain was found highly stable in terms of surface activity and EI indices at different environmental factors i.e. temperature, pH and various NaCl concentrations, where, emulsifying property was found high stable till 30 days of incubation. Moreover, the stain was capable to degrade hydrocarbon at 42.2 ± 0.04 %, and the Gas chromatography- mass spectrometry (GC-MS) profile showed the majority of peak intensities of hydrocarbons have been completely degraded compared to control.

Production and characterization of surfactin-like biosurfactant produced by novel strain Bacillus nealsonii S2MT and it's potential for oil contaminated soil remediation.

BACKGROUND: Biosurfactants, being highly biodegradable, ecofriendly and multifunctional compounds have wide applications in various industrial sectors including environmental bioremediation. Surfactin, a member of lipopeptide family, which is considered as one of the most powerful biosurfactants due to its excellent emulsifying activities as well as environmental and therapeutic applications. Therefore, the aim of this study was to investigate the newly isolated bacterial strain S2MT for production of surfactin-like biosurfactants and their potential applications for oil-contaminated soil remediation. RESULTS: In this study, the strain S2MT was isolated from lake sediment and was identified as Bacillus nealsonii based on transmitted electron microscopy (TEM) and 16S rRNA ribo-typing. The strain S2MT produced biosurfactant that reduced the surface tension (34.15 ± 0.6 mN/m) and displayed excellent emulsifying potential for kerosene (55 ± 0.3%). Additionally, the maximum biosurfactant product yield of 1300 mg/L was achieved when the composition of the culture medium was optimized through response surface methodology (RSM). Results showed that 2% glycerol and 0.1% NH4NO3 were the best carbon/nitrogen substrates for biosurfactant production. The parameters such as temperature (30 °C), pH (8), agitation (100 rpm), NH4NO3 (0.1%) and NaCl (0.5%) displayed most significant contribution towards surface tension reduction that resulted in enhanced biosurfactant yield. Moreover, the extracted biosurfactants were found to be highly stable at environmental factors such as salinity, pH and temperature variations. The biosurfactants were characterized as cyclic lipopeptides relating to surfactin-like isoforms (C13-C15) using thin-layer chromatography (TLC), Ultra high performance liquid chromatography and mass spectrometry (UHPLC-MS). The crude biosurfactant product displayed up to 43.6 ± 0.08% and 46.7 ± 0.01% remediation of heavy engine-oil contaminated soil at 10 and 40 mg/L concentrations, respectively. CONCLUSION: Present study expands the paradigm of surfactin-like biosurfactants produced by novel isolate Bacillus nealsonii S2MT for achieving efficient and environmentally acceptable soil remediation as compared to synthetic surfactants.

Enhanced Oil Recovery by Potential Biosurfactant-Producing Halo-thermotolerant Bacteria Using Soil Washing and Sand-Packed Glass Column Techniques.

Biosurfactants offer numerous advantages over the chemical surfactants, especially in energy and environment-related applications. Microbial enhanced oil recovery (MEOR) is a technique to recover oil from reservoirs by using microbes and their metabolites. In present study, total sixteen morphologically distinct bacterial strains isolated from different salty areas of the district Khairpur Mir's, Pakistan, were investigated for their MEOR potential. Screening assays for thermotolerance and halotolerance declared 7 out of 16 (43.75%) bacterial isolates as thermotolerant (capable of growing in the temperature range 60-70 °C) and halotolerant (tolerating NaCl concentrations up to 17%, w/v). Moreover, five of them were screened as biosurfactant producers. Among, the lowest surface tension reduction was achieved with biosurfactants produced by the strains KJ2MO (27.8 mN/m) and KJ2SK (29.3 mN/m). The biosurfactant activity was found stable at temperature (100-121 °C, 1 h) and pH (4-10). Moreover, maximum oil recovery was obtained with biosurfactant of bacterial strain KJ2MO (54.7%, 51.25%) followed by KJ2SK (44.7%, 40.5%), KJ1WB (37%, 35.5%) and KJ2MD (37.8%, 31.9%) by using either techniques, i.e., soil washing and sand-packed column, respectively. Moreover, the potent species were identified as Pseudomonas pseudoalcaligenes KJ1WB, Bacillus aerius KJ2MD, Bacillus licheniformis KJ2SK, and Bacillus subtilis KJ2MO using 16S rRNA ribo-typing. The investigated species were found to be promising biosurfactants producers having potential for enhanced oil recovery and could be used in other environmental applications like bioremediation.

Biosurfactants-based mixed polycyclic aromatic hydrocarbon degradation: From microbial community structure toward non-targeted metabolomic profile determination.

Biosurfactants-based bioremediation is considered an efficient technology to eliminate environmental pollutants including polycyclic aromatic hydrocarbons (PAHs). However, the precise role of rhamnolipids or lipopeptide-biosurfactants in mixed PAH dissipation, shaping microbial community structure, and influencing metabolomic profile remained unclear. In this study, results showed that the maximum PAH degradation was achieved in lipopeptide-assisted treatment (SPS), where the pyrene and phenanthrene were substantially degraded up to 74.28 % and 63.05 % respectively, as compared to rhamnolipids (SPR) and un-aided biosurfactants (SP). Furthermore, the high throughput sequencing analysis revealed a significant change in the PAH-degrading microbial community, with Proteobacteria being the predominant phylum (>98 %) followed by Bacteroidota and Firmicutes in all the treatments. Moreover, Pseudomonas and Pannonibacter were found as highly potent bacterial genera for mixed PAH degradation in SPR, SPS, and SP treatments, nevertheless, the abundance of the genus Pseudomonas was significantly enhanced (>97 %) in SPR treatment groups. On the other hand, the non-targeted metabolomic profile through UHPLC-MS/MS exhibited a remarkable change in the metabolites of amino acids, carbohydrates, and lipid metabolisms by the input of rhamnolipids or lipopeptide-biosurfactants whereas, the maximum intensities of metabolites (more than two-fold) were observed in SPR treatment. The findings of this study suggested that the aforementioned biosurfactants can play an indispensable role in mixed PAH degradation as well as seek to offer new insights into shifts in PAH-degrading microbial communities and their metabolic function, which can guide the development of more efficient and targeted strategies for complete removal of organic pollutants such as PAH from the contaminated environment.

A comprehensive study on microbial-surfactants from bioproduction scale-up toward electrokinetics remediation of environmental pollutants: Challenges and perspectives.

Currently, researchers have focused on electrokinetic (EK) bioremediation due to its potential to remove a wide-range of pollutants. Further, to improve their performance, synthetic surfactants are employed as effective additives because of their excellent solubility and mobility. Synthetic surfactants have an excessive position in industries since they are well-established, cheap, and easily available. Nevertheless, these surfactants have adverse environmental effects and could be detrimental to aquatic and terrestrial life. Owing to social and environmental awareness, there is a rising demand for bio-based surfactants in the global market, from environmental sustainability to public health, because of their excellent surface and interfacial activity, higher and stable emulsifying property, biodegradability, non- or low toxicity, better selectivity and specificity at extreme environmental conditions. Unfortunately, challenges to biosurfactants, like expensive raw materials, low yields, and purification processes, hinder their applicability to large-scale. To date, extensive research has already been conducted for production scale-up using multidisciplinary approaches. However, it is still essential to research and develop high-yielding bacteria for bioproduction through traditional and biotechnological advances to reduce production costs. Herein, this review evaluates the recent progress made on microbial-surfactants for bioproduction scale-up and provides detailed information on traditional and advanced genetic engineering approaches for cost-effective bioproduction. Furthermore, this study emphasized the role of electrokinetic (EK) bioremediation and discussed the application of BioS-mediated EK for various pollutants remediation.

Grassland ecology system: A critical reservoir and dissemination medium of antibiotic resistance in Xilingol Pasture, Inner Mongolia.

Antibiotic resistance is a major threat to human health. It is necessary to explore all the potential sources and comprehend the pathways that antibiotic resistance genes (ARGs) are transmitted. In this study, by applying high-throughput quantitative PCR and high-throughput sequencing, ARGs and microbial community structure were determined, to understand the reservoirs and spread of ARGs in the Xilingol grassland system. A total of 151,140 and 138 different ARGs were observed in manure, soil, and water samples, respectively. Only 12 ARGs were shared in all environmental and animal manure samples. Multidrug defense system, such as efflux pump, was the most dominant factor in manure and soil samples, followed by antibiotic deactivation processes. These genes coffering resistance to major classes of antibiotics including ß_Lactamase (blaSFO, fox5, blaCTX-M-04, blaOXY), vancomycin (vanC-03, vanXD), MLSB (vatE-01, mphA-01), aminoglycoside (aadA2-01), Multidrug (oprJ) and others (oprD, qacEdelta1-02), except sulfonamide and tetracycline. The 12 ARGs were significantly enriched in water samples compared to manure and soil samples (p < 0.01) and demonstrated that the water environment was an important transmission source of ARGs in the grassland. The highest enrichment was up to 324.5-fold. Moreover, the 12 shared ARGs were positively correlated with the mobile genetic elements (p < 0.01). The nonrandom co-occurrence network patterns between ARGs and microbial community suggested that a total of three bacterial phyla were viewed as the potential ARGs hosts. These findings indicate that ARGs were highly enriched in water samples, demonstrating that the water environment was a critical source and sink of ARGs in the grassland system. It may illuminate the mechanism stressing the effects of human activity on the occurrence and transmission of ARGs in the grassland system.

Acceleration of emergence of E. coli antibiotic resistance in a simulated sublethal concentration of copper and tetracycline co-contaminated environment.

An environment co-contaminated with metals and antibiotics ultimately exposes bacteria to these metals and antibiotics simultaneously. This study aims to explore the efficacy of sublethal concentrations of copper ions contaminated with tetracycline regarding antibiotic resistance in a sensitive strain of E. coli K12. The study proved that a copper ions and tetracycline co-contaminated environment could considerably enhance the mutation frequencies of chloramphenicol and polymyxin B resistance in antibiotic susceptible E. coli; however, the equivalent copper ions and tetracycline alone showed weaker effects. Results also demonstrated that an environment co-contaminated with relatively high sublethal concentrations of copper ion and tetracycline co-contaminated environment could induce much higher antibiotic resistance than the low sublethal and control groups. Whole-genome characterization results indicated that variability existed within the genotype and phenotype involved in antibiotic resistance. Additionally, the evolved resistant strains displayed hereditary resistance after 5 round culture cycles in LB broth over 5 days. Results implied that co-contamination with metals and antibiotics environment could strengthen resistance and contribute to the induction and dissemination of antibiotic resistance in metal and antibiotic co-contaminated environment.

Effect of natural microbiome and culturable biosurfactants-producing bacterial consortia of freshwater lake on petroleum-hydrocarbon degradation.

Freshwater lake ecosystem is a reservior of valuable microbial diversity. It needs to be explored for addressing key environmental issues like petroleum-hydrocarbon contamination. In this work, the microbial communities (pre and post enriched with petroleum-hydrocarbons) from different layers of freshwater lake, i.e. surface water, sediments and deepwater, were explored through metagenomic and culture-dependent approaches. A total of 41 bacterial phyla were retrieved from pre-enriched samples, which were significantly reduced in enriched samples where Proteobacteria were dominant (87% to 100%) followed by Bacteroidetes (7.37%) and Verrucomicrobia (3.06%). The most dominant hydrocarbon-degrading genera were extensively verified as Pseudomonas (48.65%), Acinetobacter (45.38%), Stenotrophomonas (3.16%) and Brevundimonas (2.07%) in surface water (S1WCC); Acinetobacter (62.46%), Aeromonas (10.7%), Sphingobacterium (5.20%) and Pseudomonas (4.23%) in sediment (S2MCC); and Acinetobacter (46.57%), Pseudomonas (13.10%), Comamonas (12.93%), Flavobacterium (12.18%) and Enterobacter (9.62%) in deep water (S4WCC). Additionally, the maximum biodegradation of petroleum-hydrocarbons (i.e. used engine oil or UEO) was achieved by microbiome of S2MCC (67.60 ± 0.08%) followed by S4WCC (59.70 ± 0.12%), whereas only 36.80 ± 0.10% degradation was achieved by S1WCC microbiome. On the other hand, UEO degradation by cultivable biosurfactant-producing single cultures such as Pseudomonas sp. S2WE, Pseudomonas sp. S2WG, Pseudomonas sp. S2MS, Ochrobactrum sp. S1MM and Bacillus nealsonii S2MT showed 31.10 ± 0.08% to 40.50 ± 0.11% biodegradation. Comparatively, the biodegradation efficiency was found higher (i.e. 42.20 ± 0.12% to 56.10 ± 0.12%) in each consortia comprising of two, three, four, and five bacterial cultures. Conclusively, the isolated culturable biosurfactants-producing bacterial consortium of freshwater lake demonstrated >80% contribution in the total petroleum-hydrocarbons degradation by the natural microbiome of the ecosystem.

Bioprospecting of rhamnolipids production and optimization by an oil-degrading Pseudomonas sp. S2WE isolated from freshwater lake.

The present study revealed biosurfactants production by a novel oil-degrading Pseudomonas sp. S2WE isolated from hydrocarbon enriched water sample, where the genus Pseudomonas (48.65%) was dominated amongst several other genera. Biosurfactants produced by this strain showed the great potential for surface tension reduction (SFT) and emulsification. The extracted crude biosurfactants were characterized using ultra-high-performance liquid chromatography-Mass Spectrometry (UHPLC-MS) and identified various mono-and di-rhamnolipids homologs from the mixture. Moreover, the lowest SFT 33.05 ± 0.1 mN/m and highest emulsification of 60.65 ± 0.64% were achieved from rhamnolipids produced from glycerol with urea. Compared to initial screening, almost (>87%) higher emulsification was observed. In addition, the biosurfactants were found highly stable at different environmental factors i.e. temperature (4 °C-121 °C), pH (3-10) and NaCl conc. (1-9%). The high stable rhamnolipids produced by new Pseudomonas sp. S2WE in this study could widely be used in enormous industrial as well as environmental applications.

Using easy-to-biodegrade co-substrate to eliminate microcystin toxic on electrochemically active bacteria and enhance bioelectricity generation from cyanobacteria biomass.

Cyanobacterial biomass is a promising natural resource for power generation, through the reactions bio-catalyzed by electrochemically active bacteria (EAB). However, the major limitation is the involvement of Microcystin-LR (MC-LR) in inhibiting EAB activation. In this work, toxic M. aeruginosa biomass was employed as analyte of a microbial fuel cell (MFC), and sodium acetate was applied as easy-to-biodegrade co-substrate to alleviate the MC-LR stress on EAB survival. The running stability was continuously enhanced with the increment of co-substrate concentration. The sufficient co-substrate supply (6.0 mM) eliminated the negative effects of MC-LR on the cyanobacteria biomass fed-MFC performance; it contributed 12.7% extension on the electric cyclic terms and caused the productions of the power density which was comparable and even 3.8% higher than its corresponding control (MFC treated with acetate alone). The co-substrate addition also increased coulombic efficiency by 60.1%, microcystin-LR removal efficiency increased by 64.7%, and diversified the microbial community with more species able to biodegrade the MC-LR, bio-transforming the metabolites and EAB. Microcystin-degrading bacteria, such as Sphingopyxis sp., Burkholderia-Paraburkholderia, and Bacillus sp., were remarkably increased, and EAB, including Shewanella sp., Desulfovibrio desulfuricans, Aeromonas hydrophila, were also much more enriched in co-substrate use protocol. Therefore, this study verified a co-substrate strategy for simultaneously eliminating MC-LR toxin and enhancing bioelectricity generation from cyanobacterial biomass via an MFC.