The metabolism of pyrene by a novel Altererythrobacter sp. with in-situ co-substrates: A mechanistic analysis based on pathway, genomics, and enzyme activity.

Using co-substrates to enhance the metabolic activity of microbes is an effective way for high-molecular-weight polycyclic aromatic hydrocarbons removal in petroleum-contaminated environments. However, the long degradation period and exhausting substrates limit the enhancement of metabolic activity. In this study, Altererythrobacter sp. N1 was screened from petroleum-contaminated soil in Shengli Oilfield, China, which could utilize pyrene as the sole carbon source and energy source. Saturated aromatic fractions and crude oils were used as in-situ co-substrates to enhance pyrene degradation. Enzyme activity was influenced by the different co-substrates. The highest degradation rate (75.98%) was achieved when crude oil was used as the substrate because strain N1 could utilize saturated and aromatic hydrocarbons from crude oil simultaneously to enhance the degrading enzyme activity. Moreover, the phthalate pathway was dominant, while the salicylate pathway was secondary. Furthermore, the Rieske-type aromatic cyclo-dioxygenase gene was annotated in the Altererythrobacter sp. N1 genome for the first time. Therefore, the co-metabolism of pyrene was sustained to achieve a long degradation period without the addition of exogenous substrates. This study is valuable as a potential method for the biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons.

Biopolymers Produced by Sphingomonas Strains and Their Potential Applications in Petroleum Production.

The genus Sphingomonas was established by Yabuuchi et al. in 1990, and has attracted much attention in recent years due to its unique ability to degrade environmental pollutants. Some Sphingomonas species can secrete high-molecular-weight extracellular polymers called sphingans, most of which are acidic heteropolysaccharides. Typical sphingans include welan gum, gellan gum, and diutan gum. Most sphingans have a typical, conserved main chain structure, and differences of side chain groups lead to different rheological characteristics, such as shear thinning, temperature or salt resistance, and viscoelasticity. In petroleum production applications, sphingans, and their structurally modified derivatives can replace partially hydrolyzed polyacrylamide (HPAM) for enhanced oil recovery (EOR) in high-temperature and high-salt reservoirs, while also being able to replace guar gum as a fracturing fluid thickener. This paper focuses on the applications of sphingans and their derivatives in EOR.

Effect of Rhamnolipid Amidation on Biosurfactant Adsorption Loss and Oil-Washing Efficiency.

Surfactant adsorption loss seriously hinders the economy of surfactant binary flooding technology for enhancing oil recovery, especially for biosurfactants with higher manufacturing costs. Here, biosurfactant rhamnolipid (RL) is chemically modified to develop a more efficient surfactant, rhamnolipid monoethanol amide (RL-MEA), which is characterized by decreased adsorption loss and increased oil-washing efficiency for enhanced oil recovery at a laboratory scale. Synthesis and characterization of the rhamnolipid monoethanol amide are carried out using high-performance liquid chromatography (HPLC), HPLC/MS, 1H nuclear magnetic resonance (NMR), and Fourier transform infrared (FTIR) spectroscopy. The aggregation behavior is disclosed by surface tension, dynamic light scattering, and fluorescence spectra with pyrene as the probe. The applied performances of RL-MEA in the simulated enhanced oil recovery are researched, including the efficiency of oil washing, wettability to crude oil, and adsorption isotherms on silicates. Compared with the critical micelle concentration (CMC) of rhamnolipid of 14.23 × 10-5 M in pure water and 9.02 × 10-5 M in 0.2 M NaCl solution, the modified RL-MEA shows a significantly lower CMC of 7.15 × 10-5 M in pure water and 5.34 × 10-5 M in 0.2 M NaCl solution. More importantly, the modified RL-MEA reduces adsorption loss by 20% and enhanced oil-washing efficiency at higher temperatures and salt concentrations compared with the parent RLs. These findings would provide valuable information for developing efficient surfactant flooding agents for harsh reservoir geological conditions.

Non-syntrophic methanogenic hydrocarbon degradation by an archaeal species.

The methanogenic degradation of oil hydrocarbons can proceed through syntrophic partnerships of hydrocarbon-degrading bacteria and methanogenic archaea1-3. However, recent culture-independent studies have suggested that the archaeon 'Candidatus Methanoliparum' alone can combine the degradation of long-chain alkanes with methanogenesis4,5. Here we cultured Ca. Methanoliparum from a subsurface oil reservoir. Molecular analyses revealed that Ca. Methanoliparum contains and overexpresses genes encoding alkyl-coenzyme M reductases and methyl-coenzyme M reductases, the marker genes for archaeal multicarbon alkane and methane metabolism. Incubation experiments with different substrates and mass spectrometric detection of coenzyme-M-bound intermediates confirm that Ca. Methanoliparum thrives not only on a variety of long-chain alkanes, but also on n-alkylcyclohexanes and n-alkylbenzenes with long n-alkyl (C≥13) moieties. By contrast, short-chain alkanes (such as ethane to octane) or aromatics with short alkyl chains (C≤12) were not consumed. The wide distribution of Ca. Methanoliparum4-6 in oil-rich environments indicates that this alkylotrophic methanogen may have a crucial role in the transformation of hydrocarbons into methane.

Production of the biosurfactant serrawettin W1 by Serratia marcescens S-1 improves hydrocarbon degradation.

With the frequent occurrence of oil spills, the bioremediation of petroleum hydrocarbons pollution has attracted more and more attention. In this study, we investigated the biodegradation of crude oil by the biosurfactant-producing strain S-1. The strain was isolated from petroleum-contaminated soil and identified as Serratia marcescens according to partial 16S rDNA gene analysis. It was able to effectively degrade hydrocarbons with the concomitant production of biosurfactants at 20-30 °C, while there was no biosurfactant production and the degradation rate was lower at 37 °C. The biosurfactant was identified as serrawettin W1 by UPLC-ESI-MS, and was found to reduce the surface tension of water to 30 mN/m, with stable surface activity and emulsion activity at temperatures from 20 to 100 °C, pH of 2-10 and NaCl concentrations of 0-50 g/L. Serrawettin W1 significantly increased the cell surface hydrophobicity (CSH) and enhanced the bioavailability of hydrocarbon pollutants, which was conducive to the degradation of crude oil, including long-chain alkanes and aromatic hydrocarbons. Serratia marcescens S-1 has potential applications in bioremediation at low temperature.

Performance Evaluation of Enzyme Breaker for Fracturing Applications under Simulated Reservoir Conditions.

Fracturing fluids are being increasingly used for viscosity development and proppant transport during hydraulic fracturing operations. Furthermore, the breaker is an important additive in fracturing fluid to extensively degrade the polymer mass after fracturing operations, thereby maximizing fracture conductivity and minimizing residual damaging materials. In this study, the efficacy of different enzyme breakers was examined in alkaline and medium-temperature reservoirs. The parameters considered were the effect of the breaker on shear resistance performance and sand-suspending performance of the fracturing fluid, its damage to the reservoir after gel breaking, and its gel-breaking efficiency. The experimental results verified that mannanase II is an enzyme breaker with excellent gel-breaking performance at medium temperatures and alkaline conditions. In addition, mannanase II did not adversely affect the shear resistance performance and sand-suspending performance of the fracturing fluid during hydraulic fracturing. For the same gel-breaking result, the concentration of mannanase II used was only one fifth of other enzyme breakers (e.g., mannanase I, galactosidase, and amylase). Moreover, the amount of residue and the particle size of the residues generated were also significantly lower than those of the ammonium persulfate breaker. Finally, we also examined the viscosity-reducing capability of mannanase II under a wide range of temperatures (104-158 °F) and pH values (7-8.5) to recommend its best-use concentrations under different fracturing conditions. The mannanase has potential for applications in low-permeability oilfield development and to maximize long-term productivity from unconventional oilwells.

Genetic engineering of the precursor supply pathway for the overproduction of the nC14-surfactin isoform with promising MEOR applications.

BACKGROUND: Surfactin, a representative biosurfactant of lipopeptide mainly produced by Bacillus subtilis, consists of a cyclic heptapeptide linked to a ß-hydroxy fatty acid chain. The functional activity of surfactin is closely related to the length and isomerism of the fatty acid chain. RESULTS: In this study, the fatty acid precursor supply pathway in Bacillus subtilis 168 for surfactin production was strengthened through two steps. Firstly, pathways competing for the precursors were eliminated with inactivation of pps and pks. Secondly, the plant medium-chain acyl-carrier protein (ACP) thioesterase (BTE) from Umbellularia californica was overexpressed. As a result, the surfactin titer after 24 h of cultivation improved by 34%, and the production rate increased from 0.112 to 0.177 g/L/h. The isoforms identified by RP-HPLC and GC-MS showed that the proportion of nC14-surfactin increased 6.4 times compared to the control strain. A comparison of further properties revealed that the product with more nC14-surfactin had higher surface activity and better performance in oil-washing. Finally, the product with more nC14-surfactin isoform had a higher hydrocarbon-emulsification index, and it increased the water-wettability of the oil-saturated silicate surface. CONCLUSION: The obtained results identified that enhancing the supply of fatty acid precursor is very essential for the synthesis of surfactin. At the same time, this study also proved that thioesterase BTE can promote the production of nC14-surfactin and experimentally demonstrated its higher surface activity and better performance in oil-washing. These results are of great significance for the MEOR application of surfactin.

Acetoin modulates conformational change of surfactin: Interfacial assembly and crude oil-washing performance.

Due to its special structure, the cyclic lipopeptide surfactin showed remarkable responsiveness to stimuli such as pH, temperature and metal ions. However, few studies investigated the effect of fermented by-products on the conformational change and interfacial assembly of surfactin. Here, the effect of acetoin, a primary metabolite of Bacillus subtilis, on the conformational change and interfacial assembly of surfactin was studied in detail. Surface tension measurements showed that the critical micelle concentration (CMC) of surfactin increased from 1.14 × 10-5 to 4.32 × 10-5 M in the presence of acetoin. Moreover, acetoin has increased the interfacial tension of surfactin aqueous solution-crude oil from 1.08 mN/m to 3.01 mN/m. Circular dichroism (CD) spectra and dynamic light-scattering (DLS) further demonstrated that acetoin had induced the conformational transition of surfactin from ß-sheet to ß-turn structure, and caused surfactin forming some larger micelle aggregations. Afterwards, it was further found that acetoin decreased the oil sand cleaning efficiency of surfactin from 59.7% to 6.6%, and deteriorated the O/W emulsion stability and altered the silicate wettability toward less water wet state. Based on the experimental results, a possible mechanism of the interaction between surfactin and acetoin was proposed.

Efficient production of surfactin from xylose-rich corncob hydrolysate using genetically modified Bacillus subtilis 168.

As one of the most powerful biosurfactants, surfactin has extensive application prospects in numerous industrial fields. Bacillus subtilis 168 was genetically modified to produce surfactin by increasing the supply of the precursor fatty acyl-CoA by overexpressing 4' phosphopantetheinyl transferase, medium-chain acyl-acyl carrier protein (ACP) thioesterase and fatty acyl CoA ligase (encoded by sfp, bte, and yhfL, respectively), and knocking out acyl-CoA dehydrogenase (encoded by fadE). The resulting recombinant strain BSFX022 produced 2203 mg/L surfactin with xylose as carbon source. The lower accumulation of organic acids with xylose as carbon source made it possible to maintain surfactin production in a non-buffered fermentation system, and the yield reached 2074 mg/L. Furthermore, to reduce the costs, waste biomass such as corncob hydrolysate and monosodium glutamate wastewater (MGW) were used, and 2032 mg/L of surfactin was produced in the optimal waste-based medium. To our best knowledge, this is the first report of surfactin production using genetically modified Bacillus subtilis 168 with xylose as carbon source.

Biodegradation of aliphatic and polycyclic aromatic hydrocarbons by the thermophilic bioemulsifier-producing Aeribacillus pallidus strain SL-1.

The utilization of thermophilic hydrocarbon-degrading microorganisms is a suitable strategy for improving biodegradation of petroleum hydrocarbons and PAHs, as well as enhancing oil recovery from high-temperature reservoirs. In this study, the thermophilic strain Aeribacillus pallidus SL-1 was evaluated for the biodegradation of crude oil and PAHs at 60 °C. Strain SL-1 was found to preferentially degrade short-chain n-alkanes (

Designer bioemulsifiers based on combinations of different polysaccharides with the novel emulsifying esterase AXE from Bacillus subtilis CICC 20034.

BACKGROUND: Bioemulsifiers are surface-active compounds, which exhibit advantages including low toxicity, higher biodegradability and biocompatibility over synthetic chemical surfactants. Despite their potential benefits, some obstacles impede the practical applications of bioemulsifiers, including low yields and high purification costs. Here, we aimed to exploit a novel protein bioemulsifier with efficient emulsifying activity and low-production cost, as well as proposed a design-bioemulsifier system that meets different requirements of industrial emulsification in the most economical way. RESULTS: The esterase AXE was first reported for its efficient emulsifying activity and had been studied for possible application as a protein bioemulsifier. AXE showed an excellent emulsification effect with different hydrophobic substrates, especially short-chain aliphatic and benzene derivatives, as well as excellent stability under extreme conditions such as high temperature (85 °C) and acidic conditions. AXE also exhibited good stability over a range of NaCl, MgSO4, and CaCl2 concentrations from 0 to 1000 mM, and the emulsifying activity even showed a slight increase at salt concentrations over 500 mM. A design-bioemulsifier system was proposed that uses AXE in combination with a variety of polysaccharides to form efficient bioemulsifier, which enhanced the emulsifying activity and further lowered the concentration of AXE needed in the complex. CONCLUSIONS: AXE showed a great application potential as a novel bioemulsifier with excellent emulsifying ability. The AXE-based-designer bioemulsifier could be obtained in the most economical way and open broad new fields for low-cost, environmentally friendly bioemulsifiers.

Biodegradation of Petroleum Hydrocarbons by Bacillus subtilis BL-27, a Strain with Weak Hydrophobicity.

The biodegradation of petroleum hydrocarbons has many potential applications and has attracted much attention recently. The hydrocarbon-degrading bacterium BL-27 was isolated from petroleum-polluted soil and was compounded with surfactants to improve biodegradation. Its 16S rDNA and rpoD gene sequences indicated that it was a strain of Bacillus subtilis. Strain BL-27 had extensive adaptability and degradability within a broad range of temperatures (25-50 °C), pH (4.0-10.0) and salinity (0-50 g/L NaCl). Under optimal conditions (45 °C, pH 7.0, 1% NaCl), the strain was able to degrade 65% of crude oil (0.3%, w/v) within 5 days using GC-MS analysis. Notably, strain BL-27 had weak cell surface hydrophobicity. The adherence rate of BL-27 to n-hexadecane was 29.6% with sucrose as carbon source and slightly increased to 33.5% with diesel oil (0.3%, w/v) as the sole carbon source, indicating that the cell surface of BL-27 is relatively hydrophilic. The strain was tolerant to SDS, Tween 80, surfactin, and rhamnolipids at a concentration of 500 mg/L. The cell surface hydrophobicity reduced more with the addition of surfactants, while the chemical dispersants, SDS (50-100 mg/L) and Tween 80 (200-500 mg/L), significantly increased the strain's ability to biodegrade, reaching 75-80%. These results indicated that BL-27 has the potential to be used for the bioremediation of hydrocarbon pollutants and could have promising applications in the petrochemical industry.

High-Yield Di-Rhamnolipid Production by Pseudomonas aeruginosa YM4 and its Potential Application in MEOR.

Rhamnolipids are a mixture of the homologs species due to variations in the rhamnose units and ß-hydroxy fatty acid moieties, mainly including Rha-C10-C10, Rha-Rha-C10-C10, and Rha-C10. In this study, strain P. aeruginosa YM4 was selected for its capacity to efficiently produce di-rhamnolipid (Rha-Rha-C10-C10) as the predominant component with soybean oil and glycerol as carbon source, accounting for 64.8% and 85.7% of total products, respectively. The critical micelle concentration (CMC) of rhamnolipid products varies with the content of di-rhamnolipid, whereby lower CMC values corresponding to higher di-rhamnolipid contents. The rhamnolipids containing 85.7% di-rhamnolipid had the lowest CMC value of 50 mg/L. Accordingly the viscosity-reducing efficiency and oil-washing efficiency of rhamnolipids increased with higher di-rhamnolipid component. At a concentration of 500 mg/L, the rhamnolipids containing 85.7% di-rhamnolipid worked best and showed 82.5% oil-washing efficiency, which offered great promise for applications in enhanced oil recovery. The results showed the variation of structure and composition of rhamnolipids had a significant effect on their application.

Genome and transcriptome analysis of Bacillus velezensis BS-37, an efficient surfactin producer from glycerol, in response to d-/l-leucine.

Surfactin is one of the most widely studied biosurfactants due to its many potential applications in different fields. In the present study, Bacillus velezensis BS-37, initially identified as a strain of Bacillus subtilis, was used to efficiently produce surfactin with the addition of glycerol, an inexpensive by-product of biodiesel production. After 36 hr of growth in glycerol medium, the total surfactin concentration reached more than 1,000 mg/L, which was two times higher than that in sucrose medium. Moreover, the addition of l- and d-Leu to the culture medium had opposite effects on surfactin production by BS-37. While surfactin production increased significantly to nearly 2,000 mg/L with the addition of 10 mM l-Leu, it was dramatically reduced to about 250 mg/L with the addition of 10 mM d-Leu. To systemically elucidate the mechanisms influencing the efficiency of this biosynthesis process, we sequenced the genome of BS-37 and analyzed changes of the transcriptome in glycerol medium in response to d-/l-leucine. The RPKM analysis of the transcriptome of BS-37 showed that the transcription levels of genes encoding modular surfactin synthase, the glycerol utilization pathway, and branched-chain amino acid (BCAA) synthesis pathways were all at a relatively high level, which may offered an explanation why this strain can efficiently use glycerol to produce surfactin with a high yield. Neither l-Leu nor d-Leu had a significant effect on the expression of genes in these pathways, indicating that l-Leu plays an important role as a precursor or substrate involved in surfactin production, while d-Leu appears to act as a competitive inhibitor. The results of the present study provide new insights into the synthesis of surfactin and ways of its regulation, and enrich the genomic and transcriptomic resources available for the construction of high-producing strains.

[Growth and metabolism of osmo-sensentive yeast Y02724 and high-osmotic resistant yeast Hansel in alginate-chitosan-alginate microcapsules].

To study the effect of the osmotic stress in the microenvironment on the growth and metabolism of the encapsulated cells under aerobic condition, Osmo-sensitive yeast Y02724 and high-osmotic resistant yeast Hansel were used as models to explore the growth and metabolism state of the cells cultivated inalginate-chitosan-alginate (ACA) microcapsules. The changes of the yeast cells' specific growth rate, maximum product quantity and the secretion of ethanol and glycerol were analyzed. For Y02724, the yield of ethanol was increased in the ACA microenvironment compared to suspension cultivation. For Hansel, the maximum growth speed of microencapsulated cultivation had no obvious difference compared to the suspension cultivation. Moreover, after encapsulation, the production of glycerol was decreased for both Y02724 and Hansel compared to suspension cultivation. In conclusion, osmotic stress existed in the ACA microcapsules and affected the growth and metabolism of the cells.

In Vitro and in Vivo characterization of alginate-chitosan-alginate artificial microcapsules for therapeutic oral delivery of live bacterial cells.

Oral administration of artificial cell microcapsules entrapping live bacterial cells is a promising approach in disease therapy. However, the current technology of microcapsules limits this approach. In this study, alginate-chitosan-alginate (ACA) microcapsules entrapping live bacterial cells were prepared with the purpose of oral delivery for therapy, and their in vitro and in vivo properties were investigated. Genetically engineered Escherichia coli DH5 were used as the model bacterial strain. ACA microcapsules remained intact and stable in simulated gastrointestinal fluid and the entrapped bacteria cells survived and grew normally. Moreover, ACA microcapsules were more stable than alginate-polylysine-alginate microcapsules in the rat gastrointestinal tract, which was attributed to the enhanced resistance of the ACA microcapsules to enzymatic digestion. Therefore, these results reinforce the potential of ACA microcapsules for the therapeutic oral delivery of live bacterial cells.