The biosynthesis of antifungal lipopeptides iturin and fengycin has attracted broad interest; however, there is a bottleneck in its low yield in wild strains. Because the key metabolic mechanisms in the lipopeptides synthesis pathway remain unclear, genetic engineering approaches are all ending up with a single or a few gene modifications. The aim of this study is to develop a systematic engineering approach to improve the antifungal activity and biosynthesis of iturin and fengycin in Bacillus amyloliquefaciens. First, blocking the carbon overflow metabolic pathway to increase precursor supply of the branched-chain amino acids by knockout of bdh, disrupting sporulation to extend the stage for producing antifungal lipopeptides by deletion of kinA, blocking of siderophore synthesis to enhance the availability of amino acids and fatty acids by deletion of dhbF, and increasing Spo0A∼P by deletion of rapA, could improve the antifungal activity by 24%, 10%, 13% and 18%, respectively. Second, the double knockout strain ΔbdhΔkinA, triple knockout strain ΔbdhΔkinAΔdhbF and quadruple knockout strain ΔkinAΔbdhΔdhbFΔrapA could improve the antifungal activity by 38%, 44% and 53%, respectively. Finally, overexpression of sfp in ΔkinAΔbdhΔdhbFΔrapA further increased the antifungal activity by 65%. After purifying iturin and fengycin as standards for quantitative analysis of lipopeptides, we found the iturin titer was 17.0 mg/L in the final engineered strain, which was 3.2-fold of the original strain. After fermentation optimization, the titer of iturin and fengycin reached 31.1 mg/L and 175.3 mg/L in flask, and 123.5 mg/L and 1200.8 mg/L in bioreactor. Compared to the original strain, the iturin and fengycin titer in bioreactor increased by 22.8-fold and 15.9-fold in the final engineered strain, respectively. This study may pave the way for the commercial production of green antifungal lipopeptides, and is also favorable for understanding the regulatory and biosynthetic mechanism of iturin and fengycin.

Tight reservoirs, as an important alternative for conventional energy resources, have been successfully exploited with the aid of hydraulic fracturing technologies. Because of the inherent ultralow permeability and porosity, tight oil reservoirs generally suffer from the effects of stress sensitivity. Both hydraulic fractures with complex geometries and a high-permeability area known as stimulated reservoir volume (SRV) may be generated by the massive hydraulic fracturing operations. All these bring huge challenges in transient pressure analysis of tight reservoirs. Up till now, although many research studies have been carried out on the transient pressure analysis of volume-fractured horizontal wells in tight reservoirs, unfortunately, there is still a lack of research studies that have taken stress sensitivity, complex fracture networks, and the SRV into consideration, simultaneously. To fill up this gap, this paper first idealizes the reservoir after hydraulic fracturing as two radial composite regions, that is, the unstimulated outer region and the inner SRV. The stress sensitivity is characterized by the variable permeability depending on the pore pressure. A linear source with consideration of the stress sensitivity in the composite reservoir is obtained by the perturbation technique, Laplace transformation, and the flow coupling of two regions. Second, the complex fracture networks are discretized into segments to capture their geometries. A semi-analytical model is finally established and validated by the comparison with previous models. On the basis of our model, six flow stages of volume-fractured horizontal well are identified and special features of each regime are analyzed. The stress sensitivity has a great impact on the later stage of production. The mobility ratio and the SRV radius mainly affect SRV pseudo-steady-state flow period and interporosity flow period in the outer region. Fracture number mainly affects the linear flow in the SRV. Fracture geometries mainly affect linear flow and interporosity flow in the SRV. This study has some significance for well test interpretation and production performance analysis of tight reservoirs.

Nowadays, unloading gas wells with coiled tubing is a common application to the field. However, it still lacks of adequate understanding of dynamic behavior of the unloading process. This paper investigates the process of liquid unloading by gas lift with coiled tubing under transient condition. This unloading process can be divided into three stages: liquid rising in tubing, liquid slug production, and liquid production by entrainment. In each stage, the mass and linear momentum conversation equations are applied as governing equations. The components of each stage includes coiled tubing, coiled tubing-tubing annulus, liquid slug, gas bubble, and liquid film. Empirical correlations have been used including surface gas injection choke, check valve, friction factor, the relationship between the gas bubble and the liquid slug velocity, inflow performance relationship, and blackoil fluid properties. From the above, the dynamic model coupling real-time change of inflow performance relationship. Using the LU factorization and Euler's method to solve the proposed dynamic model in time domain. Among all these variables, the most important ones include gas injection rates, pressures at various locations, length of the liquid slug and gas bubble, and various velocities. By the simulation efforts, the mechanism of liquid unloading process is revealed. Gas lift is commonly constrainted by gas availability. This is pioneer study on liquid unloading with coiled tubing is critical in design phase to optimize the usage of injected gas as well as choose appropriate pump and cost-saving for electricity expense.

No abstract

Trilinear flow model is an effective method to reproduce the flow behavior for horizontal wells with multistage hydraulic fracture treatments in unconventional reservoirs. However, models developed so far for transient analysis have rarely considered the inflow performance of wells. This paper introduces a new composite dual-porosity trilinear flow model for the multiple-fractured horizontal well in the naturally fractured reservoirs. The analytical solution is derived under a constant rate condition for analyzing transient pressure behaviors and generating the transient inflow performance relationships (IPRs). The plots of pressure profiles with time could provide insightful information about various flow regimes that develop throughout the entire production cycle. Sensitivity analysis of pressure and pressure derivative response was also performed by varying different parameters (such as hydraulic fracture width and permeability, reservoir configurations, etc.) and by which the impacts of different parameters on the durations of regimes as well as the productivity index can be confirmed. The main outcomes obtained from this study are as follows: (1) the ability to characterize naturally fractured reservoirs using a new composite dual-porosity trilinear flow model; (2) the application of analytical solutions of transient analysis to generate transient IPR curves for different flow regimes; (3) understanding the effect of reservoir configurations, fractures, and matrix characteristics on pressure distribution, flow regime duration, and transient IPR. More specifically, the pressure drop increases and the productivity index decreases with the decrease of the hydraulic fracture conductivity and the increase of matrix permeability and the skin factor. Also, the larger hydraulic fracture spacing and drainage area result in the later onset of the pseudo-steady-state regime. (4) A comprehensive study on transient pressure behaviors and transient inflow performance can provide valuable information to characterize the multifractured complex systems as well as some insights into the production.